AU2016341044A1 - Restoring function to a non-functional gene product via guided Cas systems and methods of use - Google Patents

Abstract

Compositions and methods are provided for restoring function to a non-functional gene product in the genome of a cell. The methods and compositions employ a guide polynucleotide /Cas endonuclease system to restore function to a non-functional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas endonucleases, sgRNAs and guide RNA/Cas complexes.

Description

BACKGROUND

Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and/or modify (edit) specific endogenous chromosomal sequences, thus altering the organism’s phenotype. Site-specific integration techniques, which employ site-specific recombination systems, as well as other types of recombination technologies, have been used to generate targeted insertions of genes of interest in a variety of organism. Genome-editing techniques such as designer zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), or homing meganucleases, are available for producing targeted genome perturbations, but these systems tends to have a low specificity and employ designed nucleases that need to be redesigned for each target site, which renders them costly and time-consuming to prepare.

Although several approaches have been developed to target a specific site for modification in the genome of an organism, there still remains a need for new

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PCT/US2016/057279 genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the genome of an organism.

BRIEF SUMMARY

Compositions and methods are provided for restoring function to a non5 functional gene product in the genome of a cell. The methods and compositions employ a guide polynucleotide /Cas endonuclease system to restore function to a non-functional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas endonucleases, guide RNAs and guide RNA/Cas complexes by introducing the components of the complex via mRNA molecules (guide RNA, Cas endonuclease) or protein molecules (Cas endonuclease) or by directly introducing the guide RNA/Cas ribonucleotide-protein complex (RGEN) itself into the cell.

In one embodiment of the disclosure, the method comprises a method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product. The disrupted gene can comprise a base pair deletion of the 4^th nucleotide upstream (5’) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional. The base pair deletion can be located at the first, second or third nucleotide of a codon sequence. The restoration can be accomplished by Non-Homologous-End -Joining (NHEJ) resulting in the insertion of

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PCT/US2016/057279 a single base at the double strand break site, or can be accomplished by the insertion of a single base at the double strand break site without the use of

Homologous Recombination or Homology Directed Repair

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein. The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene. The modification in the targeted nucleotide sequence can be selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, or a substitution of at least one nucleotide in said target site. The method can further comprise introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, or a donor DNA wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site.

In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising: a) introducing into at least one cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing

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PCT/US2016/057279 a double strand break in said nucleotide sequence; b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and, c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.

In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence in the genome of a plant without the use of a polynucleotide modification template or donor DNA, the method comprising: a) introducing into at least one plant cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a plant cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; c) regenerating a plant from the plant cell of (b); d) introducing into a cell from the plant of (c) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and inserting a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template; and, e) optimally, selecting a cell comprising the nucleotide insertion of (d).

The guide RNA and Cas endonuclease protein forming the guide RNA /Cas endonuclease complex can be introduced into the cell directly as RNA and protein, respectively, or as a ribonucleotide-protein complex. The components of the guide RNA/Cas endonuclease complex can be introduced as mRNA molecules encoding the Cas endonuclease protein and as RNA comprising the guide RNA, or as recombinant DNA molecules encoding the guide RNA and the Cas endonuclease protein.

In one embodiment of the disclosure, the method comprises a DNA free method of delivering a guide RNA /Cas endonuclease complex into a cell, the

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PCT/US2016/057279 method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotideprotein and matrix to bind and form a ribonucleotide-protein-matrix complex; and, introducing said ribonucleotide-protein-matrix complex into said cell.

In one embodiment of the disclosure, the method comprises a DNA free method of delivering guide RNA /Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said guide RNA and said Cas endonuclease protein to form a complex inside said cell.

The DNA free method can further comprise introducing a polynucleotide template, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii). The DNA free method can also further comprise introducing a donor DNA, wherein said donor DNA comprises at least one polynucleotide of interest. The introduction into the cell can be via a delivery system selected from the group consisting of particle mediated delivery, whisker mediated delivery, cell-penetrating peptide mediated delivery, electroporation, PEP-mediated transfection and nanoparticle mediated delivery.

Cells include human, non-human, animal, archaea, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell.

Also provided are nucleic acid constructs, cells, plants, progeny plants, microorganisms, explants, seeds and grain produced by the methods described herein. Additional embodiments of the methods and compositions of the present disclosure are shown herein.

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BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application. The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.821-1.825. The sequence descriptions contain the three letter codes for amino acids as defined in 37 C.F.R. §§ 1.821-1.825, which are incorporated herein by reference.

Figures io Figure 1 depicts an alignment and count of the top 10 most frequent NHEJ mutations induced by the maize optimized guide RNA/Cas endonuclease system described herein. The mutations were identified by deep sequencing. The reference sequence (SEQ ID NO: 48) represents the unmodified locus with each target site shown in bold. The PAM sequence (grey) and expected site of cleavage (arrow) are also indicated. Deletions or insertions as a result of imperfect NHEJ are shown by a or an italicized underlined nucleotide, respectively. The reference and mutations

1-10 of the target site correspond to SEQ ID NOs: 49-58, respectively. In maize, for the majority of target sites, the most prevalent type of mutation generated by Cas9gRNA system is a single nucleotide insertion (> 60%) (count shown as 16,861).

Figure 2 depicts partial nucleotide sequences ((SEQ ID NOs: 59, 61,63) and partial amino acid sequences (SEQ ID NOs: 60, 62, 64) of the ALS2 gene and two editing repair templates (Oligol and Oligo2); modified nucleotides are underlined and the codon sequence targeted for gene editing (Pro to Ser) is boxed.

Figure 3 depicts maize plants having an edited ALS2 allele for resistance to chlorsulfuron (left) and wild type plants (right). Four-week old plants were sprayed with chlorsulfuron (100 mg/L). Plants are shown three weeks after the treatment.

Figure 4A-4C shows a schematic of a fragment of the ALS2 gene (SEQ ID NOs: 65, 67, 69) selected for modification and use of ALS2 as a selectable marker. The encoded amino acid sequences are shown below each nucleotide sequence.

(SEQ ID NOs: 66, 68). Figure 4A: A single nucleotide (G) in position 165 (bold and underlined) can be removed in order to generate a specific knock-out version of the edited for chlorsulfuron resistance ALS2 gene. Figure 4B depicts the new

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PCT/US2016/057279 nucleotide sequence with a single nucleotide deletion (G removed) resulting in the translational frame shift and ALS2 gene knock-out. Figure 4C: The ALS2 gene function and chlorsulfuron resistance are restored through insertion of a single nucleotide (N, bold and underlined) during the process of DSB repair via NHEJ pathway.

Figure 5A-5B. Re-activation of inactivated ALS2P165S as selectable marker. Figure 5A (SEQ ID NO: 70). A design of ALS 2P165S gene containing upstream out-of-frame translational start codon located 3 nucleotides 5’ of PAM. Initiation of translation at the first AUG (depicted by arrow below sequence) encodes a 4 amino io acid polypeptide which prevents the initiation of translation start codon of ALS2 (grey letters). Figure 5B (SEQ ID NO: 71). Single nucleotide insertion (C, AorT) or deletion (or any combination) that results in the loss of the upstream AUG allows initiation of translation at the start codon of ALS2 (depicted by arrow below sequence) restoring translation of the full-length ALS2P165S herbicide resistance gene.

Figure 6A-6C shows a schematic of a fragment of polynucleotide of interest (SEQ ID NO: 72) comprising an endogenous target site selected for modification. The encoded amino acid sequences are shown below each nucleotide sequence. (SEQ ID NOs: 73, 75, 77). Figure 6A depicts single nucleotide (in this example C, shown in bold and underlined) located next to an endonuclease cleavage site (shown by arrow) can be removed through NHEJ. Figure 6B depicts the resulting polynucleotide of interest (SEQ ID NO:74) having a single base deleted, resulting in the creation of a new cleavage site (indicated by arrow) and translational frameshift. Figure 4C: A single nucleotide (in this example T, shown in bold and underlined) located next to an endonuclease cleavage site can be inserted through NHEJ without the use of a polynucleotide modification (repair) template, resulting in a single nucleotide edit of the polynucleotide of interest (SEQ ID NO:76). PAM sequences are highlighted in grey.

Figure 7. Top: Agrobacterium vector for stable integration of the UBI:Cas9 into the maize genome. Bottom: Agrobacterium vector for stable integration of the MDH:Cas9 into the maize genome. MDH is a temperature regulated promoter, regulating expression of the Cas9. These vectors also contain visible marker gene

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PCT/US2016/057279 (END2:AmCYAN), which was used for selection of stably transformed callus sectors. Sequence of the Red Fluorescent Protein (DsRED) contained duplicated in a direct orientation 369 bp fragments separated by a 343-bp spacer, which contained sequences for recognition and targeting by two gRNAs and LIG3:4 meganuclease. H2B refers to the histone H2B gene promoter.

Sequences

Table 1. Summary of Nucleic Acid and Protein SEQ ID Numbers

Description	Nucleic acid SEQ ID NO.	Protein SEQ ID NO.
Cas9 coding sequence	1
potato ST-LS1 intron	2
SV40 NLS		3
VirD2 NLS	4
Maize optimized Cas9 expression cassette	5
Lig-CR3 guide RNA expression vector	6
Maize genomic target site MS26Cas-1 plus PAM sequence	7
Maize genomic target site MS26Cas-2 plus PAM sequence	8
Maize genomic target site MS26Cas-3 plus PAM sequence	9
Maize genomic target site LIGCas-1 plus PAM sequence	10
Maize genomic target site LIGCas-2 plus PAM sequence	11
Maize genomic target site LIGCas-3 plus PAM sequence	12
Maize genomic target site MS45Cas-1 plus PAM sequence	13
Maize genomic target site MS45Cas-2 plus PAM sequence	14
Maize genomic target site MS45Cas-3 plus PAM sequence	15
Maize genomic target site ALSCas-1 plus PAM sequence	16
Maize genomic target site ALSCas-2 plus PAM sequence	17
Maize genomic target site ALSCas-3 plus PAM sequence	18
Primer sequences	19-38
ALS1-DNA sequence	39
ALS2-DNA sequence	40
full length Zm-ALS2 protein		41

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Maize genomic target site ALSCas-4 plus PAM sequence	42
794 bp polynucleotide modification template	43
127 bp polynucleotide modification template, oligol	44
127 bp polynucleotide modification template, oligo2	45
Agrobacterium vector containing maize codon optimized Cas9 and maize UBI promoter	46
Agrobacterium vector containing maize codon optimized Cas9 and maize MDH promoter	47
Sequences shown in Figure 1	48-58
Sequences shown in Figure 2	59, 61,63	60, 62, 64
Sequences shown in Figure 4A-4C	65, 67, 69	66,68
Sequences shown in Figure 5A-5B	70-71
Sequences shown in Figure 6A-6C	72, 74, 76	73, 75,77
IN2 promoter	78
ALSCas7 target site	79
ALSCas7-1 target site which is the modified ALSCas7 target site	80
maize off target site	81

DETAILED DESCRIPTION

Compositions and methods are provided for restoring function to a nonfunctional gene product in the genome of a cell. The methods and compositions employ a guide RNA/Cas endonuclease system to restore function to a nonfunctional gene product and to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. The present disclosure also describes methods for modifying a nucleotide sequence in the genome of a cell using a restored functional selectable marker, as well as methods for editing a nucleotide sequence in the genome a cell without introducing a polynucleotide modification template into said cell. Compositions and methods are also provided for DNA free delivery of Cas9 endonucleases, sgRNAs and guide RNA/Cas complexes by introducing the components of the complex (guide RNA and Cas endonucleases) via mRNA molecules (guide RNA, Cas endonuclease) or protein molecules (Cas endonuclease) or by directly introducing the nucleotide-protein complex into the cell.

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CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) (also known as SPIDRs-SPacer Interspersed Direct Repeats) constitute a family of DNA loci. CRISPR loci consist of short and highly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to 140 times - also referred to as CRISPR-repeats) which are partially palindromic. The repeated sequences (usually specific to a species) are interspaced by variable sequences of constant length (typically 20 to 58 by depending on the CRISPR locus (W02007/025097, published March 1,

2007). Bacteria and archaea have evolved adaptive immune defenses termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR10 associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids (W02007/025097, published March 1,2007). Multiple CRISPR-Cas systems have been described including Class 1 systems, with multisubunit effector complexes, and Class 2 systems, with single protein effectors (such as but not limiting to Cas9, Cpf1 ,C2c1,C2c2, C2c3). (Zetsche et al., 2015, Cell 163, 1-13;

Shmakov et al., 2015, Molecular_Cell 60, 1-13; Makarova et al. 2015, Nature

Reviews Microbiology Vol. 13:1-15, WO 2013/176772 A1 published on November 23, 2013 and incorporated by its entirety by reference herein).

The type II CRISPR/Cas system from bacteria employs a crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNA contains a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA (trans-activating CRISPR RNA) forming a RNA duplex that directs the Cas endonuclease to cleave the DNA target. Spacers are acquired through a not fully understood process involving Cas1 and Cas2 proteins. All type II CRISPR-Cas loci contain cas1 and cas2 genes in addition to the cas9 gene (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Cas gene includes a gene that is generally coupled, associated or close to, or in the vicinity of flanking CRISPR loci. The terms “Cas gene”, “CRISPR-associated (Cas) gene” are used interchangeably herein. A comprehensive review of the Cas protein family is presented in Haft et al.

(2005) Computational Biology, PLoS Comput Biol 1(6): e60.

doi:10.1371/journal.pcbi.0010060. As described therein, 41 CRISPR-associated (Cas) gene families are described, in addition to the four previously known gene

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PCT/US2016/057279 families. It shows that CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. The number of Cas genes at a given CRISPR locus can vary between species (Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6): e60. doi:10.1371/journal.pcbi.0010060; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; WO 2013/176772 A1 published on November 23, 2013 and incorporated by its entirety by reference herein).

The term “Cas endonuclease” herein refers to a protein encoded by a Cas (CR IS PR-associated) gene. A Cas endonuclease, when in complex with a suitable io polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific DNA target sequence. A Cas endonuclease described herein comprises one or more nuclease domains. Cas endonucleases of the disclosure include those having a HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain (Makarova et al. 2015,

Nature Reviews Microbiology Vol. 13:1-15). A Cas includes a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Casio, or complexes of these.

As used herein, the terms “guide polynucleotide/Cas endonuclease complex” “guide polynucleotide/Cas endonuclease system”, “ guide polynucleotide/Cas complex”, “guide polynucleotide/Cas system”, “guided Cas system” , “PGEN” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease protein that are capable of forming a polynucleotideprotein complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide polynucleotide/Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the four known CRISPR systems (Horvath and Barrangou, Science 327:167-170) such as a type I, II, or III CRISPR system. A Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cleaves at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in

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PCT/US2016/057279 complex with the Cas protein. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, a Cas protein herein may lack DNA cleavage or nicking activity, but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and US 2015-0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference).

A guide polynucleotide/Cas endonuclease complex can cleave one or both io strands of a DNA target sequence. A guide polynucleotide/Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprises a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Thus, a wild type Cas protein (e.g., a Cas9 protein disclosed herein), or a variant thereof retaining some or all activity in each endonuclease domain of the Cas protein, is a suitable example of a Cas endonuclease that can cleave both strands of a DNA target sequence. A Cas9 protein comprising functional RuvC and HNH nuclease domains is an example of a Cas protein that can cleave both strands of a DNA target sequence. A guide polynucleotide/Cas endonuclease complex that can cleave one strand of a DNA target sequence can be characterized herein as having nickase activity (e.g., partial cleaving capability). A Cas nickase typically comprises one functional endonuclease domain that allows the Cas to cleave only one strand (i.e., make a nick) of a DNA target sequence. For example, a Cas9 nickase may comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild type HNH domain). As another example, a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant, dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed by Gasiunas et al. (Proc. Natl. Acad. Sci. U.S.A. 109:E2579-E2586), Jinek et al. (Science 337:816-821), Sapranauskas et al. (Nucleic Acids Res. 39:92759282) and in U.S. Patent Appl. Publ. No. 2014/0189896, which are incorporated herein by reference.

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A pair of Cas9 nickases can be used to increase the specificity of DNA targeting. In general, this can be done by introducing two Cas9 nickases that, by virtue of being associated with RNA components with different guide sequences, target and nick nearby DNA sequences on opposite strands in the region for desired targeting. Such nearby cleavage of each DNA strand creates a double strand break (i.e., a DSB with single-stranded overhangs), which is then recognized as a substrate for non-homologous-end-joining, NHEJ (prone to imperfect repair leading to mutations) or homologous recombination, HR. Each nick in these embodiments can be at least about 5,10,15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 (or any integer io between 5 and 100) bases apart from each other, for example. One or two Cas9 nickase proteins herein can be used in a Cas9 nickase pair. For example, a Cas9 nickase with a mutant RuvC domain, but functioning HNH domain (i.e., Cas9 HNH+/RuvC-), could be used (e.g., Streptococcus pyogenes Cas9 HNH+/RuvC-). Each Cas9 nickase (e.g., Cas9 HNH+/RuvC-) would be directed to specific DNA sites nearby each other (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences targeting each nickase to each specific DNA site.

A Cas protein can be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1,2, 3, or more domains in addition to the Cas protein). Such a fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains, such as between Cas and a first heterologous domain. Examples of protein domains that may be fused to a Cas protein herein include, without limitation, epitope tags (e.g., histidine [His], V5,

FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]), and domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g., VP16 or VP64), transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. A Cas protein can also be

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PCT/US2016/057279 in fusion with a protein that binds DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A

DNA binding domain, and herpes simplex virus (HSV) VP16.

A Cas protein herein can be from any of the following genera: Aeropyrum,

Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus,

Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Streptococcus, Treponema, Francisella, or Thermotoga. Alternatively, a Cas protein herein can be encoded, for example, by any of SEQ ID NOs:462-465, 467-472, 47415 477, 479-487, 489-492, 494-497, 499-503, 505-508, 510-516, or 517-521 as disclosed in U.S. Appl. Publ. No. 2010/0093617, which is incorporated herein by reference.

A guide polynucleotide/Cas endonuclease complex in certain embodiments can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence. Such a complex may comprise a Cas protein in which all of its nuclease domains are mutant, dysfunctional. For example, a Cas9 protein herein that can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence, may comprise both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain. A Cas protein herein that binds, but does not cleave, a target DNA sequence can be used to modulate gene expression, for example, in which case the Cas protein could be fused with a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein).

The Cas endonuclease gene can be a Type II Cas9 endonuclease , such as but not limited to, Cas9 genes listed in SEQ ID NOs: 462, 474, 489, 494, 499, 505, and 518 of W02007/025097published March 1,2007, and incorporated herein by reference. In another embodiment, the Cas endonuclease gene is a plant, maize or

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PCT/US2016/057279 soybean optimized Cas9 endonuclease gene. The Cas endonuclease gene herein can be a plant or microbial codon optimized Cas9 endonuclease gene. The Cas endonuclease gene can be operably linked to a SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region.

“Cas9” (formerly referred to as Cas5, Csn1, or Csx12) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically io recognizing and cleaving all or part of a DNA target sequence. A Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157:1262-1278). A type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans20 activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA.

The amino acid sequence of a Cas9 protein described herein, as well as certain other Cas proteins herein, may be derived from a Streptococcus (e.g., S. pyogenes, S. pneumoniae, S. thermophilus, S. agalactiae, S. parasanguinis, S.

oralis, S. salivarius, S. macacae, S. dysgalactiae, S. anginosus, S. constellatus, S. pseudoporcinus, S. mutans), Listeria (e.g., L. innocua), Spiroplasma (e.g., S. apis,

S. syrphidicola), Peptostreptococcaceae, Atopobium, Porphyromonas (e.g., P. catoniae), Prevotella (e.g., P. intermedia), Veillonella, Treponema (e.g., T. socranskii, T denticola), Capnocytophaga, Finegoldia (e.g., F. magna),

Coriobacteriaceae (e.g., C. bacterium), Olsenella (e.g., O. profusa), Haemophilus (e.g., /-/. sputorum, H. pittmaniae), Pasteurella (e.g., P. bettyae), Olivibacter (e.g., O. sifiensis), Epilithonimonas (e.g., E. tenax), Mesonia (e.g., M. mobilis), Lactobacillus

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PCT/US2016/057279 (e.g., L. plantarum), Bacillus (e.g., B. cereus), Aquimarina (e.g., A. muelleri),

Chryseobacterium (e.g., C. palustre), Bacteroides (e.g., B. graminisolvens),

Neisseria (e.g., N. meningitidis), Francisella (e.g., F. novicida), or Flavobacterium (e.g., F. frigidarium, F. soli) species, for example. As another example, a Cas9 protein can be any of the Cas9 proteins disclosed in Chylinski et al. (RNA Biology 10:726-737 and US patent application 62/162377, filed May 15, 2015 ), which are incorporated herein by reference.

Accordingly, the sequence of a Cas9 protein herein can comprise, for example, any of the Cas9 amino acid sequences disclosed in GenBank Accession io Nos. G3ECR1 (S. thermophilus), WP_026709422, WP_027202655, WP_027318179, WP_027347504, WP_027376815, WP_027414302, WP_027821588, WP_027886314, WP_027963583, WP_028123848, WP_028298935, Q03JI6 (S. thermophilus), EGP66723, EGS38969, EGV05092, EHI65578 (S. pseudoporcinus), EIC75614 (S. oralis), EID22027 (S. constellatus),

EIJ69711, EJP22331 (S. oralis), EJP26004 (S. anginosus), EJP30321, EPZ44001 (S. pyogenes), EPZ46028 (S. pyogenes), EQL78043 (S. pyogenes), EQL78548 (S. pyogenes), ERL10511, ERL12345, ERL19088 (S. pyogenes), ESA57807 (S. pyogenes), ESA59254 (S. pyogenes), ESU85303 (S. pyogenes), ETS96804, UC75522, EGR87316 (S. dysgalactiae), EGS33732, EGV01468 (S. oralis),

EHJ52063 (S. macacae), EID26207 (S. oralis), EID33364, EIG27013 (S.

parasanguinis), EJF37476, EJO19166 (Streptococcus sp. BS35b), EJU16049, EJU32481, YP_006298249, ERF61304, ERK04546, ETJ95568 (S. agalactiae), TS89875, ETS90967 (Streptococcus sp. SR4), ETS92439, EUB27844 (Streptococcus sp. BS21), AFJ08616, EUC82735 (Streptococcus sp. CM6),

EWC92088, EWC94390, EJP25691, YP_008027038, YP_008868573, AGM26527,

AHK22391, AHB36273, Q927P4, G3ECR1, or Q99ZW2 (S. pyogenes), which are incorporated by reference. A variant of any of these Cas9 protein sequences may be used, but should have specific binding activity, and optionally endonucleolytic activity, toward DNA when associated with an RNA component herein. Such a variant may comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

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96%, 97%, 98%, or 99% identical to the amino acid sequence of the reference

Cas9.

Alternatively, a Cas9 protein herein can be encoded by any of SEQ ID NOs:462 (S. thermophilus), 474 (S. thermophilus), 489 (S. agalactiae), 494 (S.

agalactiae), 499 (S. mutans), 505 (S. pyogenes), or 518 (S. pyogenes) as disclosed in U.S. Appl. Publ. No. 2010/0093617 (incorporated herein by reference), for example. Alternatively still, a Cas9 protein may comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the io foregoing amino acid sequences, for example. Such a variant Cas9 protein should have specific binding activity, and optionally cleavage or nicking activity, toward DNA when associated with an RNA component herein.

A Cas protein herein such as a Cas9 can comprise a heterologous nuclear localization sequence (NLS). A heterologous NLS amino acid sequence herein may be of sufficient strength to drive accumulation of a Cas protein in a detectable amount in the nucleus of a yeast cell herein, for example. An NLS may comprise one (monopartite) or more (e.g., bipartite) short sequences (e.g., 2 to 20 residues) of basic, positively charged residues (e.g., lysine and/or arginine), and can be located anywhere in a Cas amino acid sequence but such that it is exposed on the protein surface. An NLS may be operably linked to the N-terminus or C-terminus of a Cas protein herein, for example. Two or more NLS sequences can be linked to a Cas protein, for example, such as on both the N- and C-termini of a Cas protein. Non-limiting examples of suitable NLS sequences herein include those disclosed in U.S. Patent Nos. 6660830 and 7309576 (e.g., Table 1 therein), which are both incorporated herein by reference.

The Cas endonuclease can comprise a modified form of the Cas9 polypeptide. The modified form of the Cas9 polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas9 protein. For example, in some instances, the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide (US patent application

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US20140068797 A1, published on March 6, 2014). In some cases, the modified form of the Cas9 polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas9” or “deactivated cas9 (dCas9).” Catalytically inactivated Cas9 variants include Cas9 variants that contain mutations in the HNH and RuvC nuclease domains. These catalytically inactivated Cas9 variants are capable of interacting with sgRNA and binding to the target site in vivo but cannot cleave either strand of the target DNA.

A catalytically inactive Cas9 can be fused to a heterologous sequence (US patent application US20140068797 A1, published on March 6, 2014). Suitable io fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA. Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Further suitable fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription regulator, etc.). A catalytically inactive Cas9 can also be fused to a Fokl nuclease to generate double strand breaks (Guilinger et al. Nature biotechnology, volume 32, number 6, June 2014).

The terms “functional fragment “, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a Cas endonuclease are used interchangeably herein, and refer to a portion or subsequence of a Cas endonuclease sequence in which the ability to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break in) the target site is retained.

The terms “functional variant “, “Variant that is functionally equivalent” and “functionally equivalent variant” of a Cas endonuclease are used interchangeably herein, and refer to a variant of a Cas endonuclease in which the ability to

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PCT/US2016/057279 recognize, bind to, and optionally nick or cleave (introduce a single or double strand break in) the target site is retained. Fragments and variants can be obtained via methods such as site-directed mutagenesis and synthetic construction.

The Cas endonuclease gene includes a plant codon optimized

Streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the form N(12-30)NGG can in principle be targeted or a Cas9 endonuclease originated from an organism selected from the group consisting of Brevibacillus laterosporus, Lactobacillus reuteri Mlc3, Lactobacillus rossiae DSM 15814, Pediococcus pentosaceus SL4, Lactobacillus nodensis JCM 14932, io Sulfurospirillum sp. SCADC, Bifidobacterium thermophilum DSM 20210, Loktanella vestfoldensis, Sphingomonas sanxanigenens NX02, Epilithonimonas tenax DSM 16811, Sporocytophaga myxococcoides and Psychroflexus torquis ATCC 700755, wherein said Cas9 endonuclease can form a guide RNA/Cas endonuclease complex capable of recognizing, binding to, and optionally nicking or cleaving all or part of a DNA target sequence. Other Cas endonuclease systems have been described in US patent applications 62/162,377 filed May 15, 2015 and 62/162,353 filed May 15, 2015, both applications incorporated herein by reference.

Cas9 endonucleases can be used for targeted genome editing (via simplex and multiplex double-strand breaks and nicks) and targeted genome regulation (via tethering of epigenetic effector domains to either the Cas9 or sgRNA. Cas9 might also be engineered to function as an RNA-guided recombinase, and via RNA tethers could serve as a scaffold for the assembly of multiprotein and nucleic acid complexes (Mali et al. 2013 Nature Methods Vol. 10: 957-963.).

As used herein, the term “guide polynucleotide”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize, bind to, and optionally cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence (referred to as guide RNA, gRNA), a DNA sequence, or a combination thereof (a RNA-DNA combination sequence). Optionally, the guide polynucleotide can comprise at least one nucleotide, phosphodiester bond or linkage modification such as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine, 2’-Fluoro A, 2’-Fluoro

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U, 2'-0-Methyl RNA, phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5’ to 3’ covalent linkage resulting in circularization. A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a “guide RNA” or “gRNA” (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and US 2015-0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference).

The guide polynucleotide can be a double molecule (also referred to as duplex guide polynucleotide) comprising a crNucleotide sequence and a io tracrNucleotide sequence. The crNucleotide includes a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a second nucleotide sequence (also referred to as a tracr mate sequence) that is part of a Cas endonuclease recognition (CER) domain. The tracr mate sequence can hybridized to a tracrNucleotide along a region of complementarity and together form the Cas endonuclease recognition domain or CER domain. The CER domain is capable of interacting with a Cas endonuclease polypeptide. The crNucleotide and the tracrNucleotide of the duplex guide polynucleotide can be RNA, DNA, and/or RNA-DNA- combination sequences. In some embodiments, the crNucleotide molecule of the duplex guide polynucleotide is referred to as “crDNA” (when composed of a contiguous stretch of DNA nucleotides) or “crRNA” (when composed of a contiguous stretch of RNA nucleotides), or “crDNA-RNA” (when composed of a combination of DNA and RNA nucleotides). The crNucleotide can comprise a fragment of the crRNA naturally occurring in Bacteria and Archaea. The size of the fragment of the crRNA naturally occurring in Bacteria and Archaea that can be present in a crNucleotide disclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments the tracrNucleotide is referred to as “tracrRNA” (when composed of a contiguous stretch of RNA nucleotides) or “tracrDNA” (when composed of a contiguous stretch of DNA nucleotides) or “tracrDNA-RNA” (when composed of a combination of DNA and RNA nucleotides. In one embodiment, the RNA that guides the RNA/ Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA.

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The tracrRNA (trans-activating CRISPR RNA) contains, in the 5’-to-3’ direction, (i) a sequence that anneals with the repeat region of CRISPR type II crRNA and (ii) a stem loop-containing portion (Deltcheva et al., Nature 471:602-607). The duplex guide polynucleotide can form a complex with a Cas endonuclease, wherein said guide polynucleotide/Cas endonuclease complex (also referred to as a guide polynucleotide/Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) into the target site. (See also U.S. Patent Application US 2015-0082478 A1, published on March io 19, 2015 and US 2015-0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference.)

The guide polynucleotide can also be a single molecule (also referred to as single guide polynucleotide) comprising a crNucleotide sequence linked to a tracrNucleotide sequence. The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a Cas endonuclease recognition domain (CER domain), that interacts with a Cas endonuclease polypeptide. By “domain” it is meant a contiguous stretch of nucleotides that can be RNA, DNA, and/or RNA-DNA-combination sequence. The

VT domain and /or the CER domain of a single guide polynucleotide can comprise a RNA sequence, a DNA sequence, or a RNA-DNA-combination sequence. The single guide polynucleotide being comprised of sequences from the crNucleotide and the tracrNucleotide may be referred to as “single guide RNA” (when composed of a contiguous stretch of RNA nucleotides) or “single guide DNA” (when composed of a contiguous stretch of DNA nucleotides) or “single guide RNA-DNA” (when composed of a combination of RNA and DNA nucleotides). The single guide polynucleotide can form a complex with a Cas endonuclease, wherein said guide polynucleotide/Cas endonuclease complex (also referred to as a guide polynucleotide/Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the target site. (See also U.S. Patent Application US 2015-0082478 A1, published on March 19,

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2015 and US 2015-0059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference.)

The term “variable targeting domain” or “VT domain” is used interchangeably herein and includes a nucleotide sequence that can hybridize (is complementary) to one strand (nucleotide sequence) of a double strand DNA target site. The % complementation between the first nucleotide sequence domain (VT domain ) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,

72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,

86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or

100%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19,

20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, a RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” (of a guide polynucleotide) is used interchangeably herein and includes a nucleotide sequence that interacts with a Cas endonuclease polypeptide. A CER domain comprises a tracrNucleotide mate sequence followed by a tracrNucleotide sequence. The CER domain can be composed of a DNA sequence, a RNA sequence, a modified DNA sequence, a modified RNA sequence (see for example US 2015-0059010 A1, published on February 26, 2015, incorporated in its entirety by reference herein), or any combination thereof.

The nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can comprise a RNA sequence, a DNA sequence, or a RNA-DNA combination sequence. In one embodiment, the nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can beat least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45,

46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

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89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can comprise a tetraloop sequence, such as, but not limiting to a GAAA tetraloop sequence.

Nucleotide sequence modification of the guide polynucleotide, VT domain and/or CER domain can be selected from, but not limited to , the group consisting of a 5' cap, a 3' polyadenylated tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide poly nucleotide to a subcellular location, a modification or io sequence that provides for tracking , a modification or sequence that provides a binding site for proteins , a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2’-Fluoro A nucleotide, a 2’-Fluoro U nucleotide; a 2'-O-Methyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5’ to 3’ covalent linkage, or any combination thereof. These modifications can result in at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group of a modified or regulated stability, a subcellular targeting, tracking, a fluorescent label, a binding site for a protein or protein complex, modified binding affinity to complementary target sequence, modified resistance to cellular degradation, and increased cellular permeability.

The terms “functional fragment “, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a guide RNA, crRNA or tracrRNA are used interchangeably herein, and refer to a portion or subsequence of the guide RNA, crRNA or tracrRNA , respectively, in which the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is retained.

The terms “functional variant “, “Variant that is functionally equivalent” and “functionally equivalent variant” of a guide RNA, crRNA or tracrRNA (respectively) are used interchangeably herein, and refer to a variant of the guide RNA, crRNA or tracrRNA, respectively, in which the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is retained.

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The terms “single guide RNA, “gRNA” and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating

CRISPR RNA). The single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site.

The terms “guide RNA/Cas endonuclease complex”, “guide RNA/Cas endonuclease system”, “ guide RNA/Cas complex”, “guide RNA/Cas system”, “gRNA/Cas complex”, “gRNA/Cas system”, “RNA-guided endonuclease” , “RGEN” are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease protein that are capable of forming a complex , wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide RNA/Cas endonuclease complex herein can comprise Cas protein(s) and suitable RNA component(s) of any of the known CRISPR systems (Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular_Cell 60, 1-13; Makarova etal. 2015, Nature Reviews Microbiology Vol. 13:1-15; Horvath and Barrangou, Science 327:167-170) such as a type I, II, or III CRISPR system. A guide RNA/Cas endonuclease complex can comprise a Type II Cas9 endonuclease and at least one

RNA component (e.g., a crRNA and tracrRNA, or a gRNA). (See also U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015 and US 20150059010 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference).

The Cas endonuclease can be introduced into a cell (provided to a cell) by any method known in the art, for example, but not limited to transient introduction methods, transfection, microinjection, and/or topical application or indirectly via recombination constructs. Plant cells differ from human and animal cells in that plant

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PCT/US2016/057279 cells contain a plant cell wall which may act as a barrier to the direct delivery of the

Cas9 endonuclease into the plant cell. Recombinant DNA constructs encoding a

Cas9 endonuclease have been successfully introduced into plant cells (Svitashev et al., Plant Physiology, 2015, Vol. 169, pp. 931-945) to allow for genome editing at a target site. One possible disadvantage of stably introducing recombinant DNA constructs in plant cells is that the continued presence of Cas9 endonucleases may increase off-target effects.

As described herein, direct delivery of the Cas endonuclease into plant cells can be achieved through particle mediated delivery. Based on the experiments io described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whisker mediated delivery, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering the Cas9 endonuclease in plant cells.

Direct delivery of the Cas endonuclease (also referred to as DNA free delivery off the Cas endonuclease ) can be achieved by introducing the Cas protein, the mRNA encoding the Cas endonuclease, and/ or the RNA guided endonuclease ribonucleotide-protein complex (RGEN) itself (as a ribonucleotide-protein complex), into a cell using any method known in the art. Direct delivery of the Cas endonuclease, either via mRNA encoding the Cas endonuclease or via a polypeptide molecule is also referred to herein as DNA free delivery of the Cas endonuclease, as no DNA molecule is involved in the production of the Cas endonuclease protein. Similarly, direct delivery of the guide RNA as an RNA molecule is also referred to herein as DNA free delivery of the guide RNA. Similarly, direct delivery of the guide RNA/endonuclease complex itself (RGEN) as a ribonucleotide-protein complex, is also referred to herein as DNA free delivery of the RGEN.

Directly introducing the Cas endonuclease as a protein, or as an mRNA molecule together with a gRNA, or as a RGEN ribonucleotide-protein itself, allows for genome editing at the target site followed by rapid degradation of the RGEN

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PCT/US2016/057279 complex, and only a transient presence of the complex in the cell which leads to reduced off-target effects (as described in Example 12).

Direct delivery of these components can be accompanied by direct delivery (co-delivery) of other mRNAs that can promote the enrichment and/or visualization of cells receiving the RGEN components. For example, delivery of mRNAs encoding screenable visual markers such as fluorescence proteins (for example but not limited to Red, green, yellow, blue or combinations thereof) can also be used in lieu of, or coupled with, direct selection of a repaired disrupted, non-functional gene product.

io Described herein are methods to restore the function of a non-functional gene product by restoring the nucleotide sequence of a disrupted gene such that the restored nucleotide sequence encodes the functional gene product.

A disrupted gene refers to a gene that has been modified (disrupted) such that its gene product loses its function (referred to as a non-functional gene product) or has a reduced function when compared to the product of the corresponding gene that does not have the disruption (also referred to as the undisrupted gene). For example, a gene encoding for a functional polypeptide or protein can be disrupted (modified) such that the translation product of the disrupted gene results in a polypeptide that has lost its function or has a reduced function.

A functional gene product includes a functional protein or polypeptide that has a biological or non-biological function.

A non-functional gene product includes reference to the gene product of a disrupted gene. The non-functional gene product includes polypeptides that have lost their function (absent function) or have a reduced function when compared to the gene product of the corresponding undisrupted gene.

In one embodiment of the disclosure, the method comprises a method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional

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PCT/US2016/057279 gene product. The disrupted gene can comprise a base pair deletion of the 4^th nucleotide upstream (5’) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional. The base pair deletion can be the first, second or third nucleotide of a codon sequence. The restoration can be accomplished by Non-Homologous-End -Joining (NHEJ) resulting in the insertion of a single base at the double strand break site, or can be accomplished by the insertion of a single base at the double strand break site without the use of io Homologous Recombination or Homology Directed Repair.

Coincident with the restoration of gene function by NHEJ (through for example delivery of RGEN components or the RGEN complex itself to a cell), modification of other targets can be accomplished by the simultaneous addition of other guide-polynucleotides. Such other targets (other than the target for restoration of gene function by NHEJ) can be any target in the genome including a transgenic locus. The approach of simultaneous delivery of two or more gRNAs when one gRNA targets and activates a selectable marker through NHEJ, (such as but not limiting to conferring herbicide tolerance) and the other gRNA(s) promote DSB(s) at target site(s) different than the selectable marker (or other disrupted gene design ) and can facilitate either targeted mutagenesis, deletion, gene editing, or site-specific trait gene insertions can allow for completely transient targeted genome modifications as all other necessary components (Cas9, gRNAs) can be delivered in a form of protein and/or in vitro transcribed RNA molecules.

A disrupted gene includes reference to a marker gene (such as, but not limited to, a phenotypic marker gene and a selectable marker gene) that has been modified (disrupted) such that its gene product loses its function (for example, in case of a herbicide disrupted selectable marker gene, the disrupted gene does not confer herbicide resistance anymore).

A selectable marker and screenable marker are used interchangeably herein and includes reference to a DNA segment (such as a selectable marker gene) that allows one to identify, or select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as,

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PCT/US2016/057279 but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. A selectable marker further includes a gene that when modified or knocked-out generates a property in a cell that allows one to identify, or select for (or against) a cell that contains said property.

In one aspect, the selectable marker allows for the selection of cells by applying selection schemes, in which for example, a selective agent (such as, but not limited to an antibiotic or herbicide) is used to inhibit or kill cells or tissues that do not comprise the selectable marker, and the cells or tissues that comprise the selectable marker continue to grow due to expression of the selectable marker gene.

In one aspect, the selectable marker allows for the visual selection of cells by applying selection schemes, in which for example, a visible marker (such as a fluorescent molecule) is used to select cells that comprise the visible marker.

Selectable marker genes include, but are not limited to, chlorosulfuron resistance genes, phosphomannose isomerase genes (PMI), bialaphos resistance genes (BAR), phosphinothricin acetyltransferase (PAT) genes, hygromycin resistance genes (NPTII), glyphosate resistance genes, DNA segments that comprise restriction enzyme sites; DNA segments that encode products which provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT)); DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products which can be readily identified (referred to as visible maker genes. Visible marker genes include reference to fluorescent markers genes, such as red fluorescent marker genes, blue fluorescent marker genes, green fluorescent marker genes, yellow fluorescent marker genes), genes encoding DsRED, RFP, red fluorescent protein, CFP, GFP, green fluorescent protein) and genes encoding phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins. Selectable marker genes further include the generation of new primer sites

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PCT/US2016/057279 for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification.

Additional selectable markers include genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See for example, Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA io 89:6314-8; Yao et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol Microbiol 6:241922; Hu et al., (1987) Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; Figge et al., (1988) Cell 52:713-22; Deuschle et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-4; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-53; Deuschle et al., (1990) Science 248:480-3; Gossen, (1993) Ph.D. Thesis, University of

Heidelberg; Reinesetal., (1993) Proc. Natl. Acad. Sci. USA 90:1917-21; Labow et al., (1990) Mol Cell Biol 10:3343-56; Zambretti et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-6; Bairn et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-6; Wyborski et al., (1991) Nucleic Acids Res 19:4647-53; Hillen and Wissman, (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al., (1991) Antimicrob Agents Chemother

35:1591-5; Kleinschnidt et al., (1988) Biochemistry 27:1094-104; Bonin, (1993)

Ph.D. Thesis, University of Heidelberg; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother 36:913-9;

Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (SpringerVerlag, Berlin); Gill et al., (1988) Nature 334:721-4.

Phenotypic marker genes include genes encoding a screenable or selectable marker that includes visual markers, whether it is a positive or negative selectable marker. Any phenotypic marker can be used.

As described herein, a phenotypic and selectable marker gene can be modified to be introduced into plant cells as a disrupted gene encoding a non30 functional gene product and used as targets by double strand break inducing endonucleases for restoration back to the non-disrupted gene encoding a functional gene product, by guide RNA introduction and DNA repair.

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Phenotypic or selectable marker genes to be disrupted can be marker genes that were previously introduced into the cell and are stably incorporated into the genome of the cell. Such pre-integrated selectable marker genes can also be complemented with other genes, for example, cell developmental enhancing genes (ZmODP2 and ZmWUS, see for example PCT/US16/49144, filed August 26, 2016 and PCT/US16/49128 filed August 26, 2016, incorporated herein by reference).

As described herein, the phenotypic and selectable marker genes can be modified to be introduced into plant cells as disrupted genes (inactive forms) and used as targets by double strand break inducing endonucleases for restoration (re10 activation) by guide RNA introduction and repair.

Described herein are expression markers genes (such as but not limiting to ALS, EPSPS, BAR) that confers resistance to a compound or allows the endogenous or previously integrated marker gene or its gene product to be used as a marker, that can be modified into inactive forms and then used as targets for re15 activation by guide RNA introduction and repair as described herein (see for Example 3-4).

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by or based upon said functional selectable marker protein . The selection can include providing a chemical to the cell (such as a plant cell, or plants derived from said plant cell) expressing said functional selectable marker protein, wherein said functional selectable marker

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PCT/US2016/057279 protein makes the cell (such as a plant cell, or plants derived from said plant cell) resistant to said chemical. The chemical can be provided during any developmental stage of the cell (in case of a plant cell during any stage of plant cell or plant development) to select for the cells or organisms comprising the desired modification in their genome. The selection also includes screening of cells (such as a plant cell, or plants derived from said plant cell) for the presence of a visual marker that results from the expression of said functional selectable marker protein .

For example, as described herein, the method can be a method for modifying a nucleotide sequence in the genome of a cell, the method comprising: introducing into at least one cell comprising a target site and a disrupted resistant-ALS marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted resistant- ALS marker gene, wherein said disrupted resistance-ALS marker gene is restored without the use of a polynucleotide modification template to a non-disrupted resistant ALSmarker gene capable of encoding a functional resistance-ALS protein that confers resistance to chlorsulfuron, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by or based upon said resistance-ALS protein that confers resistance to chlorsulfuron, The selection step can include providing chlorsulfuron to the plant cell or plants derived from said plant cell at any stage during plant cell or plant development to select for the plant cell or plants comprising the desired modification the their genome.

The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene. The modification in the targeted nucleotide sequence can be selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, or a substitution of at least one nucleotide in said target site. The method can further

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PCT/US2016/057279 comprise introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, or a donor DNA wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site.

Any guided endonuclease can be used in the methods disclosed herein.

Such endonucleases include, but are not limited to Cas and Cpf1 endonucleases. Many endonucleases have been described to date that can recognize specific PAM sequences (see for example -US patent applications 62/162377 filed May 15, 2015 and 62/162353 filed May 15, 2015 and Zetsche B et al. 2015. Cell 163, 1013) and io cleave the target DNA at a specific positions. It is understood that based on the methods and embodiments described herein utilizing a guided Cas system one can now tailor these methods such that they can utilize any guided endonuclease system. For example, one can envision adapting the method for restoring function to a non-functional gene product in the genome of a cell described herein to a method comprising introducing a guided Cpf1 endonuclease complex instead of a guided Cas endonuclease complex to restore a disrupted gene and creating a functional gene product. Other guided endonucleases and nucleotide-protein complexes that find use in the methods disclosed herein include those described in WO 2013/088446.

Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, and include restriction endonucleases that cleave DNA at specific sites without damaging the bases. Restriction endonucleases include Type I, Type II, Type III, and Type IV endonucleases, which further include subtypes. In the Type I and Type III systems, both the methylase and restriction activities are contained in a single complex. Endonucleases also include meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT/US12/30061, filed on March 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs, the families are the

LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases

WO 2017/070032

PCT/US2016/057279 are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI- for enzymes encoded by free-standing

ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. This cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples the recombinase io is from the Integrase or Resolvase families.

TAL effector nucleases are a new class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. (Miller et al. (2011) Nature Biotechnology 29:143-148). Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a doublestrand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example having a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a nonspecific endonuclease domain, for example nuclease domain from a Type Ils endonuclease such as Fokl. Additional functionalities can be fused to the zincfinger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3 finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.

DNA double strand break (DSB) technologies (ZFNs, TALENs and CRISPRCas) have wide-ranging applications in academic research, gene therapy, and

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PCT/US2016/057279 animal and plant breeding programs. These technologies have been successfully used to introduce genome modifications in multiple plant species, including major crops such as maize, wheat, soybean and rice. Plant genome editing is limited by current transformation and gene modification methods, efficiency of DNA delivery, and low frequencies of plant regeneration. In contrast to human and animal systems, the presence of a thick wall surrounding every plant cell fundamentally impacts plant transformation and plant gene modification protocols. This cell wall makes it impossible to use transfection or electroporation, which are broadly used for nucleic acid and/or protein delivery in mammalian genome editing experiments.

For this reason, plant transformation and plant genome modification primarily relies on Agrobacterium-med\ated and biolistic delivery (ballistic delivery) of guide RNA/Cas endonuclease reagents on DNA vectors. As a result, gRNA and Cas9 expression cassettes frequently integrate into the genome and potentially lead to gene disruption, plant mosaicism, and potential off-site cutting. Although these undesired secondary changes can be segregated away by several rounds of backcrossing to the wild type parent plant, this process can be time consuming especially for crops with complex polyploid genomes and long breeding cycles such as, but not limited to, soybean and wheat. As described herein, delivery of Cas endonuclease and gRNAs in the form of RGEN complexes into plant cells can mitigate many of these side effects (Example 11-12). An unexpected high frequency of NHEJ-mediated mutagenesis facilitated by delivery of RGEN complexes in plants is described in Example 10. Given this high frequency of mutagenesis using a RGEN complex, DNA-free and selectable marker-free gene modification may become a practical approach to generate gene knock-outs. This DNA- and selectable marker-free approach might be less practical for gene editing and gene insertion (as compared to gene mutations by NHEJ) applications due to the low frequency of the HDR pathway in somatic plant cells. Moreover, DNA molecules often integrate into the targeted DSB sites, decreasing the efficiency of gene editing, and especially, gene insertion. It has been demonstrated that DNA vectors encoding for genes delivered into the plant cell (for example, Cas9, gRNA, selectable marker genes and trait gene) have tendency to co-integrate into the same DSB site dramatically reducing frequency of usable events with site-specific trait gene

WO 2017/070032

PCT/US2016/057279 insertions. Limiting delivered DNA molecules to donor DNA (for example, trait gene with homology arms) can increase the probability of events with desirable genotype. Therefore, the concept, described herein, of disrupted (inactive) endogenous or preintegrated selectable marker genes that can be activated upon RGEN delivery, can make the DNA- and selectable marker-free approach for gene editing and gene insertion become very practical.

The guide polynucleotide can be introduced into a cell directly, as single stranded polynucleotide or a double stranded polynucleotide, using any method known in the art such as, but not limited to, particle bombardment, whiskers mediated transformation, Agrobacterium transformation or topical applications. The guide RNA can also be introduced indirectly into a cell by introducing a recombinant DNA molecule (via methods such as, but not limited to, particle bombardment or Agrobacterium transformation) comprising a heterologous nucleic acid fragment encoding a guide RNA, operably linked to a specific promoter that is capable of transcribing the guide RNA in said cell. The specific promoter can be, but is not limited to, a RNA polymerase III promoter, which allow for transcription of RNA with precisely defined, unmodified, 5’- and 3’-ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161). As described herein, direct delivery of a sgRNA into plant cells can be achieved through particle mediated delivery. Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering gRNA in plant cells.

The guide polynucleotide can be produced by any method known in the art, including chemically synthesizing guide polynucleotides (such as but not limiting to Hendel et al. 2015, Nature Biotechnology 33, 985-989), in vitro generated guide polynucleotides, and/or self-splicing guide RNAs (such as but not limiting to Xie et al. 2015, PNAS 112:3570-3575).

The terms “target site”, “target sequence”, “target site sequence, ’’target DNA”, “target locus”, “genomic target site”, “genomic target sequence”, “genomic target

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PCT/US2016/057279 locus” and “protospacer”, are used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a transgenic locus, or any other DNA molecule in the genome (including chromosomal, choloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave . The target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms “endogenous target sequence” and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non15 conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein. An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genome of a cell but be located in a different position (i.e., a non-endogenous or non-native position) in the genome of a cell.

An “altered target site”, “altered target sequence”, “modified target site”, “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such “alterations” include, for example:

(i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).

The length of the target DNA sequence (target site) can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22,

23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The nick/cleavage site

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PCT/US2016/057279 can be within the target sequence or the nick/cleavage site could be outside of the target sequence. In another variation, the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other Cases, the incisions could be staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5' overhangs, or 3' overhangs. Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given target site, wherein the active variants retain biological activity and hence are capable of being recognized io and cleaved by an Cas endonuclease. Assays to measure the single or doublestrand break of a target site by an endonuclease are known in the art and generally measure the overall activity and specificity of the agent on DNA substrates containing recognition sites.

A “protospacer adjacent motif” (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide/Cas endonuclease system. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.

The terms “targeting”, “gene targeting” and “DNA targeting” are used interchangeably herein. DNA targeting herein may be the specific introduction of a knock-out, edit, or knock-in at a particular DNA sequence, such as in a chromosome or plasmid of a cell. In general, DNA targeting can be performed herein by cleaving one or both strands at a specific DNA sequence in a cell with a Cas protein associated with a suitable polynucleotide component. Such DNA cleavage, if a double-strand break (DSB), can prompt NHEJ or HDR processes which can lead to modifications at the target site.

The terms “knock-out”, “gene knock-out” and “genetic knock-out” are used interchangeably herein. A knock-out represents a DNA sequence of a cell that has been rendered partially or completely inoperative by targeting with a Cas protein;

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PCT/US2016/057279 such a DNA sequence prior to knock-out could have encoded an amino acid sequence, or could have had a regulatory function (e.g., promoter), for example. A knock-out may be produced by an indel (insertion or deletion of nucleotide bases in a target DNA sequence through NHEJ), or by specific removal of sequence that reduces or completely destroys the function of sequence at or near the targeting site.

The guide polynucleotide/Cas endonuclease system can be used in combination with a co-delivered polynucleotide modification template to allow for editing (modification) of a genomic nucleotide sequence of interest. (See also U.S.

Patent Application US 2015-0082478 A1, published on March 19, 2015 and

WO2015/026886 A1, published on February 26, 2015, both are hereby incorporated in its entirety by reference.)

A “modified nucleotide” or “edited nucleotide” refers to a nucleotide sequence of interest that comprises at least one alteration when compared to its non-modified nucleotide sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).

The term “polynucleotide modification template” includes a polynucleotide that comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited. A nucleotide modification can be at least one nucleotide substitution, addition or deletion. Optionally, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

A polynucleotide modification template can be introduced into a cell by any method known in the art, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct delivery.

The polynucleotide modification template can be introduced into a cell as a single stranded polynucleotide molecule, a double stranded polynucleotide

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PCT/US2016/057279 molecule, or as part of a circular DNA (vector DNA). The polynucleotide modification template can also be tethered to the guide RNA and/or the Cas endonuclease. Tethered DNAs can allow for co-localizing target and template DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10 : 957-963.) The polynucleotide modification template may be present transiently in the cell or it can be introduced via a viral replicon.

The nucleotide to be edited can be located within or outside a target site io recognized and cleaved by a Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification at a target site recognized and cleaved by a Cas endonuclease. In another embodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between the at least one nucleotide to be edited and the genomic target site.

Genome editing can be accomplished using any method of gene editing available. For example, gene editing can be accomplished through the introduction into a host cell of a polynucleotide modification template (sometimes also referred to as a gene repair oligonucleotide) containing a targeted modification to a gene within the genome of the host cell. The polynucleotide modification template for use in such methods can be either single-stranded or double-stranded. Examples of such methods are generally described, for example, in US Publication No. 2013/0019349.

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell, the method comprising:

introducing into at least one cell comprising a target site and a disrupted selectable marker gene, at least one polynucleotide modification template, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said polynucleotide modification

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PCT/US2016/057279 template comprises at least one nucleotide modification of said nucleotide sequence, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein.. The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene.

Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering a polynucleotide modification template in plant cells.

In some embodiments, gene editing may be facilitated through the induction of a double-stranded break (DSB) in a defined position in the genome near the desired alteration. DSBs can be induced using any DSB-inducing agent available, including, but not limited to, TALENs, meganucleases, zinc finger nucleases, nucleic acid guided-endonuclease systems, e.g. Cas9-gRNA systems (based on bacterial CRISPR-Cas systems), and the like. In some embodiments, the introduction of a DSB can be combined with the introduction of a polynucleotide modification template.

The process for editing a genomic sequence combining DSB and modification templates generally comprises: introducing to a host cell, a DSBinducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal

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PCT/US2016/057279 region flanking the DSB. Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015, WO2015/026886 A1, published on February 26, 2015, US application 62/023246, filed on July 07, 2014, and US application 62/036,652, filed on August 13, 2014, all of which are incorporated by reference herein.

Described herein are methods to method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template.

io Gene editing using guided Cas endonucleases systems and a polynucleotide modification templates to modify a target sequence or a nucleotide of interest near a target sequence relies on homologous recombination I homologous directed repair (HDR) mechanisms that occur at a lower frequency in plant cell when compared to the frequency of Non Homologous End Joining (NHEJ). It would be desirable to increase the frequency of gene editing by not having to rely on HDR type mechanisms. Described herein are methods that enable gene editing without the use of a polynucleotide modification template, by relying on the restoration of a disrupted gene, wherein the restoration is accomplished by Non-Homologous-End Joining (NHEJ) resulting in the insertion of at least a single base at the double strand break site or wherein the restoration is accomplished by the insertion of at least a single base at the double strand break site without the use of Homologous Recombination or Homology Directed Repair.

In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising: a) introducing into at least one cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and, c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex

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PCT/US2016/057279 capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.

In one embodiment of the disclosure, the method comprises a method for for editing a nucleotide sequence in the genome of a plant without the use of a polynucleotide modification template or donor DNA, the method comprising: a) introducing into at least one plant cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence; b) selecting a plant cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; c) regenerating a plant from the plant cell of (b); d) introducing into a cell from the plant of (c) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and inserting a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template; and, e) optimally, selecting a cell comprising the nucleotide insertion of (d).

The terms “knock-in”, “gene knock-in , “gene insertion” and “genetic knock-in” are used interchangeably herein. A knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with a Cas protein (by HR, wherein a suitable donor DNA polynucleotide is also used). Examples of knock-ins are a specific insertion of a heterologous amino acid coding sequence in a coding region of a gene, or a specific insertion of a transcriptional regulatory element in a genetic locus.

Various methods and compositions can be employed to obtain a cell or organism having a polynucleotide of interest inserted in a target site for a Cas endonuclease. Such methods can employ homologous recombination to provide integration of the polynucleotide of Interest at the target site. In one method provided, a polynucleotide of interest is provided to the organism cell in a donor DNA construct.

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As used herein, “donor DNA” includes reference to a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease. The donor DNA construct can further comprise a first and a second region of homology that flank the polynucleotide of Interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome. The donor DNA can be tethered to the guide polynucleotide and /or the Cas endonuclease. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10 : 957-963.)

By “homology” is meant DNA sequences that are similar. For example, a “region of homology to a genomic region” that is found on the donor DNA is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome. A region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site. For example, the region of homology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5- 50, 5-55, 5-60, 5-65, 5- 70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 520 300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 51400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 52400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region. “Sufficient homology” indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. The structural similarity includes overall length of each polynucleotide fragment, as well as the sequence similarity of the polynucleotides. Sequence similarity can be described by the percent sequence identity over the whole length of the sequences, and/or by conserved regions comprising localized similarities such as contiguous nucleotides having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences.

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The amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp,

450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp,

1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. The amount of homology can also io described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook etal., (1989)

Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,

NY); Current Protocols in Molecular Biology, Ausubel etal., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology2.5 Hybridization with Nucleic Acid Probes, (Elsevier, New York).

As used herein, a “genomic region” is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises a portion of the target site. The genomic region can comprise at least 510, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5- 50, 5-55, 5-60, 5-65, 5- 70, 5-75, 530 80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900,

5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900,

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5-3000, 5-3100 or more bases such that the genomic region has sufficient homology to undergo homologous recombination with the corresponding region of homology.

Polynucleotides of interest and/or traits can be stacked together in a complex trait locus as described in US-2013-0263324-A1, published 03 Oct 2013 and in

PCT/US13/22891, published January 24, 2013, both applications are hereby incorporated by reference. The guide polynucleotide/Cas9 endonuclease system described herein provides for an efficient system to generate double strand breaks and allows for traits to be stacked in a complex trait locus.

The guide polynucleotide/Cas endonuclease system can be used for io introducing one or more polynucleotides of interest or one or more traits of interest into one or more target sites by introducing one or more guide polynucleotides, one Cas endonuclease, and optionally one or more donor DNAs to a plant cell, ((as described in US patent application US-2015-0082478-A1, published on March 19, 2015, incorporated by reference herein). A fertile plant can be produced from that plant cell that comprises an alteration at said one or more target sites, wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii). Plants comprising these altered target sites can be crossed with plants comprising at least one gene or trait of interest in the same complex trait locus, thereby further stacking traits in said complex trait locus (see also US-2013-0263324-A1, published October 3, 2013 and in PCT/US13/22891, published January 24, 2013, , incorporated by reference herein).

The structural similarity between a given genomic region and the corresponding region of homology found on the donor DNA can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of homology or sequence identity shared by the “region of homology” of the donor DNA and the “genomic region” of the organism genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination

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The region of homology on the donor DNA can have homology to any sequence flanking the target site. While in some embodiments the regions of homology share significant sequence homology to the genomic sequence immediately flanking the target site, it is recognized that the regions of homology can be designed to have sufficient homology to regions that may be further 5' or 3' to the target site. In still other embodiments, the regions of homology can also have homology with a fragment of the target site along with downstream genomic regions. In one embodiment, the first region of homology further comprises a first fragment of the target site and the second region of homology comprises a second fragment of io the target site, wherein the first and second fragments are dissimilar.

Once a double-strand break is induced in the DNA, the cell’s DNA repair mechanism is activated to repair the break. The Non-Homologous-End-Joining (NHEJ) pathways are the most common repair mechanism to bring the broken ends together (Bleuyard et al., (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible. The two ends of one double-strand break are the most prevalent substrates of NHEJ (Kirik et al., (2000) EMBO J 19:5562-6), however if two different double-strand breaks occur, the free ends from different breaks can be ligated and result in chromosomal deletions (Siebert and Puchta, (2002) Plant Cell 14:1121-31), or chromosomal translocations between different chromosomes (Pacher et al., (2007) Genetics 175:21-9). Error-prone DNA repair mechanisms can produce mutations at double-strand break sites. The NonHomologous-End-Joining (NHEJ) pathways are the most common repair mechanism to bring the broken ends together (Bleuyard et al., (2006) DNA Repair

5:1-12).

Alternatively, the double-strand break can be repaired by homologous recombination (HR) between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and/or

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PCT/US2016/057279 epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81). Episomal DNA molecules can also be ligated into the double-strand break, for example, integration of T-DNAs into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol

133:956-65; Salomon and Puchta, (1998) EMBO J 17:6086-95).

As used herein, “homologous recombination (HR)” includes the exchange of

DNA fragments between two DNA molecules at the sites of homology. The frequency of homologous recombination is influenced by a number of factors. Different organisms vary with respect to the amount of homologous recombination io and the relative proportion of homologous to non-homologous recombination.

Generally, the length of the region of homology affects the frequency of homologous recombination events: the longer the region of homology, the greater the frequency. The length of the homology region needed to observe homologous recombination is also species-variable. In many cases, at least 5 kb of homology has been utilized, but homologous recombination has been observed with as little as 25-50 bp of homology. See, for example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. Natl. Acad. Sci. USA 82:476872, Sugawara and Haber, (1992) Mol Cell Biol 12:563-75, Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA

83:5199-203; Liskay et al., (1987) Genetics 115:161-7.

Homology-directed repair (HDR) is a mechanism in cells to repair doublestranded and single stranded DNA breaks. Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211).The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirements between the donor and acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR. Homology-directed repair at nicks (single-stranded breaks) can occur via a mechanism distinct from HDR at double-strand breaks (Davis and Maizels. PNAS (0027-8424), 111 (10), p. E924E932.

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Alteration of the genome of a plant cell, for example, through homologyrirected repair (HDR),, is a powerful tool for genetic engineering. Despite the low frequency of homologous recombination in higher plants, there are a few examples of successful homologous recombination of plant endogenous genes. The parameters for homologous recombination in plants have primarily been investigated by rescuing introduced truncated selectable marker genes. In these experiments, the homologous DNA fragments were typically between 0.3 kb to 2 kb. Observed frequencies for homologous recombination were on the order of 10'⁴ to IO’⁵. See, for example, Halfter et al., (1992) Mol Gen Genet 231:186-93; Offringa et io al., (1990) EMBO J 9:3077-84; Offringa et al., (1993) Proc. Natl. Acad. Sci. USA 90:7346-50; Paszkowski et al., (1988) EMBO J 7:4021-6; Hourda and Paszkowski, (1994) Mol Gen Genet 243:106-11; and Risseeuw et al., (1995) Plant J 7:109-19.

DNA double-strand breaks appear to be an effective factor to stimulate homologous recombination pathways (Puchta et al., (1995) Plant Mol Biol 28:28115 92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot

56:1-14). Using DNA-breaking agents, a two- to nine-fold increase of homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al., (1995) Plant Mol Biol 28:281-92). In maize protoplasts, experiments with linear DNA molecules demonstrated enhanced homologous recombination between plasmids (Lyznik et al., (1991) Mol Gen Genet 230:209-18).

The donor DNA may be introduced by any means known in the art. For example, a plant having a target site is provided. The donor DNA may be provided by any delivery method known in the art including, for example, Agrobacterium2.5 mediated transformation, whiskers mediated transformation, or biolistic particle bombardment. The donor DNA may be present transiently in the cell or it can be introduced via a viral replicon. In the presence of the Cas endonuclease and the target site, the donor DNA is inserted into the plant’s genome.

As described herein, direct delivery of a donor DNA into plant cells can be achieved through particle mediated delivery. Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to

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PCT/US2016/057279 protoplasts, electroporation, particle bombardment, whiskers mediated delivery, cellpenetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering a donor DNA in plant cells.

In one embodiment of the disclosure, the method comprises a method for modifying a nucleotide sequence in the genome of a cell ,the method comprising: introducing into at least one cell comprising a target site and a disrupted selectable marker gene, at least one donor DNA, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a nondisrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein . The introducing and selection step does not comprise the introduction of a selectable marker gene, such as a recombinant DNA construct comprising a selectable marker gene. The disrupted selectable marker gene can be any disrupted marker gene including a disrupted visible marker gene.

Further uses for guide RNA/Cas endonuclease systems have been described (See U.S. Patent Application US 2015-0082478 A1, published on March 19, 2015,

WO2015/026886 A1, published on February 26, 2015, US 2015-0059010 A1, published on February 26, 2015, US application 62/023246, filed on July 07, 2014, and US application 62/036,652, filed on August 13, 2014, all of which are incorporated by reference herein) and include but are not limited to modifying or replacing nucleotide sequences of interest (such as a regulatory elements), insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and/or introducing alternate splicing sites, modifications of

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PCT/US2016/057279 nucleotide sequences encoding a protein of interest, amino acid and/or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.

Polynucleotides of interest are further described herein and include polynucleotides reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for genetic engineering will change accordingly.

Further provided are methods for identifying at least one plant cell, comprising in its genome, a polynucleotide of interest integrated at the target site. A variety of methods are available for identifying those plant cells with insertion into the genome at or near to the target site without using a screenable marker phenotype. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof. See, for example, US Patent Application 12/147,834, herein incorporated by reference to the extent necessary for the methods described herein. The method also comprises recovering a plant from the plant cell comprising a polynucleotide of

Interest integrated into its genome. The plant may be sterile or fertile. It is recognized that any polynucleotide of interest can be provided, integrated into the plant genome at the target site, and expressed in a plant.

Polynucleotides/polypeptides of interest include, but are not limited to, herbicide-resistance coding sequences, insecticidal coding sequences, nematicidal coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic and biotic stress tolerance coding sequences, or sequences modifying plant traits such as yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and/or utilization, fatty acids, and oil content and/or composition. More specific polynucleotides of interest include, but are not limited to, genes that improve crop yield, polypeptides that improve desirability of crops, genes encoding proteins conferring resistance to abiotic stress, such as drought, nitrogen, temperature, salinity, toxic metals or trace elements, or those

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PCT/US2016/057279 conferring resistance to toxins such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, fertility or sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like that can be stacked or used in combination with other traits, such as but not limited to herbicide resistance, described herein.

Agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, introducing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated by reference.

Polynucleotide sequences of interest may encode proteins involved in introducing disease or pest resistance. By disease resistance or pest resistance is intended that the plants avoid the harmful symptoms that are the outcome of the plant-pathogen interactions. Pest resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Disease resistance and insect resistance genes such as lysozymes or cecropins for antibacterial protection, or proteins such as defensins, glucanases or chitinases for antifungal protection, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects are all examples of useful gene products. Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Patent No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell

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78:1089); and the like. Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and

Geiser et al. (1986) Gene 48:109); and the like.

An herbicide resistance protein or a protein resulting from expression of an herbicide resistance-encoding nucleic acid molecule includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide io for a longer period of time than cells that do not express the protein. Herbicide resistance traits may be introduced into plants by genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS also called AHAS), in particular the sulfonylurea-type (UK: sulphonylurea) herbicides, genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene and the GAT gene), HPPD inhibitors (e.g, the HPPD gene) or other such genes known in the art. See, for example, US Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and US Provisional Application No. 61/401,456, each of which is herein incorporated by reference. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.

As used herein, a “sulfonylurea-tolerant polypeptide” comprises any polypeptide which when expressed in a plant confers tolerance to at least one sulfonylurea. Sulfonylurea herbicides inhibit growth of higher plants by blocking acetolactate synthase (ALS), also known as, acetohydroxy acid synthase (AHAS). Plants containing particular mutations in ALS (e.g., the S4 and/or HRA mutations) are tolerant to sulfonylurea herbicides. The production of sulfonylurea-tolerant plants is described more fully in U.S. Patent Nos. 5,605,011; 5,013,659; 5,141,870;

5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824;

and international publication WO 96/33270, which are incorporated herein by reference in their entireties for all purposes, and in Tan et al. 2005. Imidazolinone52

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PCT/US2016/057279 tolerant crops: history, current status and future. Pest Manag Sci 61:246-257. The sulfonylurea-tolerant polypeptide can be encoded by, for example, the SuRA or SuRB locus of ALS. In specific embodiments, the ALS inhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALS mutant, the S4 mutant or the S4/HRA mutant or any combination thereof. Different mutations in ALS are known to confer tolerance to different herbicides and groups (and/or subgroups) of herbicides; see, e.g., Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Patent No. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which is herein incorporated by reference in their entirety. The HRA mutation in ALS finds particular io use in one embodiment. The mutation results in the production of an acetolactate synthase polypeptide which is resistant to at least one sulfonylurea compound in

A gene encoding a sulfonylurea-tolerant polypeptide is referred to as a sulfonyl tolerant gene or a sulfonyl resistant gene. The terms sulfonyl tolerant gene or sulfonyl resistant gene are used interchangeably herein.

A disrupted sulfonylurea resistant (ALS) gene refers to a disrupted gene, of which its corresponding undisrupted gene encodes a sulfonylurea-tolerant polypeptide, that is modified such that its gene product no longer encodes a functional sulfonylurea-tolerant polypeptide.

In one embodiment, the method comprises a method for producing a sulfonylurea resistant plant comprising a modified target site, the method comprising: a) introducing into a plant cell comprising a disrupted sulfonylurea resistant (ALS) gene, a first guide RNA, a Cas9 endonuclease, at least a second guide RNA, wherein said first guide RNA and Cas9 endonuclease can form a first complex capable of introducing a double strand in said disrupted sulfonylurea resistant (ALS) gene, wherein said second guide RNA and Cas9 endonuclease can form a second complex capable of introducing a double strand break at said target site; and, b) obtaining a sulfonylurea resistant plant from said plant cell, wherein said sulfonylurea resistant plant comprises a modification at said target, wherein said modification is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii).

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Components of a sulfonylurea-responsive repressor system (as described in US 8,257,956, issued on September 4, 2012) can also be introduced into plant genomes to generate a repressor/operator/inducer systems into said plant where polypeptides can specifically bind to an operator, wherein the specific binding is regulated by a sulfonylurea compound.

Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male fertility genes such as MS26 (see for example U.S. Patents 7,098,388, 7,517,975, 7,612,251), MS45 (see for example U.S. Patents 5,478,369, 6,265,640) io or MSCA1 (see for example U.S. Patent 7,919,676). Maize plants (Zea mays L.) can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. It can self-pollinate (“selfing”) or cross pollinate. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears. Pollination may be readily controlled by techniques known to those of skill in the art. The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selections are two of the breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines or various broadbased sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. A hybrid maize variety is the cross of two such inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential. The hybrid progeny of the first generation is designated F1. The F1 hybrid is more vigorous than its inbred parents. This hybrid vigor, or heterosis, can be manifested in many ways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility system incorporating manual detasseling. To produce hybrid seed, the male tassel is removed from the growing female inbred parent, which can be planted in various alternating row

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PCT/US2016/057279 patterns with the male inbred parent. Consequently, introducing that there is sufficient isolation from sources of foreign maize pollen, the ears of the female inbred will be fertilized only with pollen from the male inbred. The resulting seed is therefore hybrid (F1) and will form hybrid plants.

Field variation impacting plant development can result in plants tasseling after manual detasseling of the female parent is completed. Or, a female inbred plant tassel may not be completely removed during the detasseling process. In any event, the result is that the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being harvested along with the hybrid seed which is normally produced. Female inbred seed does not exhibit heterosis and therefore is not as productive as F1 seed. In addition, the presence of female inbred seed can represent a germplasm security risk for the company producing the hybrid.

Alternatively, the female inbred can be mechanically detasseled by machine.

Mechanical detasseling is approximately as reliable as hand detasseling, but is faster and less costly. However, most detasseling machines produce more damage to the plants than hand detasseling. Thus, no form of detasseling is presently entirely satisfactory, and a need continues to exist for alternatives which further reduce production costs and to eliminate self-pollination of the female parent in the production of hybrid seed.

Mutations that cause male sterility in plants have the potential to be useful in methods for hybrid seed production for crop plants such as maize and can lower production costs by eliminating the need for the labor-intensive removal of male flowers (also known as de-tasseling) from the maternal parent plants used as a hybrid parent. Mutations that cause male sterility in maize have been produced by a variety of methods such as X-rays or UV-irradiations, chemical treatments, or transposable element insertions (ms23, ms25, ms26, ms32) (Chaubal et al. (2000) Am J Bot 87:1193-1201). Conditional regulation of fertility genes through fertility/sterility “molecular switches” could enhance the options for designing new male-sterility systems for crop improvement (Unger et al. (2002) Transgenic Res 11:455-465).

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Furthermore, it is recognized that the polynucleotide of interest may also comprise antisense sequences complementary to at least a portion of the messenger RNA (mRNA) for a targeted gene sequence of interest. Antisense nucleotides are constructed to hybridize with the corresponding mRNA.

Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having 70%, 80%, or 85% sequence identity to the corresponding antisense sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene.

io Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.

In addition, the polynucleotide of interest may also be used in the sense orientation to suppress the expression of endogenous genes in plants. Methods for suppressing gene expression in plants using polynucleotides in the sense orientation are known in the art. The methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, generally greater than about 65% sequence identity, about 85% sequence identity, or greater than about 95% sequence identity. See, U.S. Patent Nos. 5,283,184 and 5,034,323; herein incorporated by reference.

The polynucleotide of interest can also be a phenotypic marker.

The recombinant DNA molecules, DNA sequences of interest, and polynucleotides of interest can comprise one or more DNA sequences for gene silencing. Methods for gene silencing involving the expression of DNA sequences in plant are known in the art include, but are not limited to, cosuppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA (hpRNA) interference, intron-containing hairpin RNA (ihpRNA) interference, transcriptional gene silencing, and micro RNA (miRNA) interference

As used herein, “nucleic acid” means a polynucleotide and includes a single or a double-stranded polymer of deoxyribonucleotide or ribonucleotide bases.

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Nucleic acids may also include fragments and modified nucleotides. Thus, the terms “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence” and “nucleic acid fragment” are used interchangeably to denote a polymer of RNA and/or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides (usually found in their 5’-monophosphate form) are referred to by their single letter designation as follows: “A” for adenosine or deoxyadenosine (for RNA or DNA, respectively), “C” for cytosine or deoxycytosine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

“Open reading frame” is abbreviated ORF.

The terms “subfragment that is functionally equivalent” and “functionally equivalent subfragment” are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of genes to produce the desired phenotype in a transformed plant. Genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence. The term “conserved domain” or “motif” means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential to the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or “signatures”, to determine if a protein with a newly determined sequence belongs to a previously identified protein family.

Polynucleotide and polypeptide sequences, variants thereof, and the structural relationships of these sequences can be described by the terms “homology”, “homologous”, “substantially identical”, “substantially similar” and

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PCT/US2016/057279 “corresponding substantially” which are used interchangeably herein. These refer to polypeptide or nucleic acid fragments wherein changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as the ability to mediate gene expression or to produce a certain phenotype. These terms also refer to modification(s) of nucleic acid fragments that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. These modifications include deletion, substitution, and/or insertion of one or more nucleotides in the nucleic acid fragment.

Substantially similar nucleic acid sequences encompassed may be defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5X SSC, 0.1 % SDS, 60°C) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Posthybridization washes determine stringency conditions.

The term selectively hybridizes includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.

The term stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence in an in vitro hybridization assay. Stringent conditions are sequencedependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).

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Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salt(s)) at pH 7.0 to 8.3, and at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% io SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.5X to 1X SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C.

“Sequence identity” or “identity” in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

The term “percentage of sequence identity” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage from 50%

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PCT/US2016/057279 to 100%. These identities can be determined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” io will mean any set of values or parameters that originally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WIND0W=5 and DIAGONALS

SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WIND0W=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

The “Clustal W method of alignment” corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition Weight=0.5,

Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB ). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

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Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: % identity and % similarity for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using a GAP creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). GAP uses the algorithm of Needleman and Wunsch, (1970) J Mol Biol 48:443-53, to find an alignment of two io complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps, using a gap creation penalty and a gap extension penalty in units of matched bases.

“BLAST” is a searching algorithm provided by the National Center for

Biotechnology Information (NCBI) used to find regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches to identify sequences having sufficient similarity to a query sequence such that the similarity would not be predicted to have occurred randomly. BLAST reports the identified sequences and their local alignment to the query sequence.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or modified naturally or synthetically wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage from 50% to 100%. Indeed, any integer amino acid identity from 50% to 100% may be useful in describing the present disclosure, such as 51%, 52%, 53%,

54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,

68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98% or 99%.

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PCT/US2016/057279 “Gene” includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences.

A “mutated gene” is a gene that has been altered through human intervention. Such a “mutated gene” has a sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion, or substitution. In certain embodiments of the disclosure, the mutated gene io comprises an alteration that results from a guide polynucleotide/Cas endonuclease system as disclosed herein. A mutated plant is a plant comprising a mutated gene.

As used herein, a “targeted mutation” is a mutation in a native gene that was made by altering a target sequence within the native gene using a method involving a double-strand-break-inducing agent that is capable of inducing a double-strand break in the DNA of the target sequence as disclosed herein or known in the art.

The guide RNA/Cas endonuclease induced targeted mutation can occur in a nucleotide sequence that is located within or outside a genomic target site that is recognized and cleaved by a Cas endonuclease.

The term “genome” as it applies to a plant cells encompasses not only 20 chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.

A “codon-modified gene” or “codon-preferred gene” or “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

An “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, that plant is heterozygous at that locus.

“Coding sequence” refers to a polynucleotide sequence which codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5’ non-coding sequences), within, or downstream (3’

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PCT/US2016/057279 non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to: promoters, translation leader sequences, 5’ untranslated sequences, 3’ untranslated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

A plant-optimized nucleotide sequence is nucleotide sequence that has been optimized for increased expression in plants. For example, a plant-optimized nucleotide sequence can be synthesized by modifying a nucleotide sequence io encoding a protein such as, for example, double-strand-break-inducing agent (e.g., an endonuclease) as disclosed herein, using one or more plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage.

Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Additional sequence modifications are known to enhance gene expression in a plant host. These include, for example, elimination of: one or more sequences encoding spurious polyadenylation signals, one or more exon-intron splice site signals, one or more transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given plant host, as calculated by reference to known genes expressed in the host plant cell. When possible, the sequence is modified to avoid one or more predicted hairpin secondary mRNA structures. Thus, a plant-optimized nucleotide sequence of the present disclosure comprises one or more of such sequence modifications.

A promoter is a region of DNA involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. An “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters

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PCT/US2016/057279 may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, and/or comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.

It has been shown that certain promoters are able to direct RNA synthesis at a higher rate than others. These are called “strong promoters”. Certain other promoters have been shown to direct RNA synthesis at higher levels only in particular types of cells or tissues and are often referred to as “tissue specific promoters”, or “tissue-preferred promoters” if the promoters direct RNA synthesis preferably in certain tissues but also in other tissues at reduced levels. Since patterns of expression of a chimeric gene (or genes) introduced into a plant are controlled using promoters, there is an ongoing interest in the isolation of novel promoters which are capable of controlling the expression of a chimeric gene or (genes) at certain levels in specific tissue types or at specific plant developmental stages.

A plant promoter can include a promoter capable of initiating transcription in a plant cell, for a review of plant promoters, see, Potenza etal., (2004) In Vitro Cell Dev Biol 40:1 -22. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al., (1985)

Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) Plant Mol Biol 12:619-32; Christensen et al., (1992) Plant Mol Biol 18:675-89); pEMU (Last etal., (1991) Theor Appl Genef 81:581-8); MAS (Velten etal., (1984) EMBO J 3:2723-30); ALS promoter (U.S. Patent No.

5,659,026), and the like. Other constitutive promoters are described in, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;

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5,399,680; 5,268,463; 5,608,142 and 6,177,611. In some examples an inducible promoter may be used. Pathogen-inducible promoters induced following infection by a pathogen include, but are not limited to those regulating expression of PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. The promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, io but are not limited to, the maize ln2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-ll-27, WO93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Other chemical-regulated promoters include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter (Schena etal., (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis eta/., (1998) Plant J 14:247-257); tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Patent Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Tissue-preferred promoters include, for example, Kawamata etal., (1997) Plant Cell Physiol 38:792-803; Hansen et al., (1997) Mol Gen Genet254:337-43; Russell etal., (1997) Transgenic Res 6:157-68; Rinehart et al., (1996) Plant Physiol 112:1331 -41; Van Camp et al., (1996) Plant Physiol

112:525-35; Canevascini et al., (1996) Plant Physiol 112:513-524; Lam, (1994)

Results Probl Cell Differ 20:181-96; and Guevara-Garcia et al., (1993) Plant J 4:495-505. Leaf-preferred promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon etal., (1994) Plant Physiol 105:357-67; Yamamoto etal., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson etal., (1958) EMBO J 4:2723-9; Timko etal., (1988) Nature 318:57-8. Root-preferred promoters include, for example, Hire et al., (1992)

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Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al., (1991) Plant Cell 3.11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root5 specific promoter of A. tumefaciens mannopine synthase (MAS)); Bogusz et al., (1990) Plant Cell 2:633-41 (root-specific promoters isolated from Parasponia andersonii and Trema tomentosa)', Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A. rhizogenes rolC and rolD root-inducing genes); Teeri et al., (1989) EMBO J 8:34350 (Agrobacterium wound-induced TR1' and TR2’ genes); VfENOD-GRP3 gene io promoter (Kuster et al., (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana etal., (1994) Plant Mol Biol 25:681-91; phaseolin gene (Murai etal., (1983) Science 23:476-82; Sengopta-Gopalen etal., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4). See also, U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363;

5,459,252; 5,401,836; 5,110,732 and 5,023,179.

Seed-preferred promoters include both seed-specific promoters active during seed development, as well as seed-germinating promoters active during seed germination. See, Thompson etal., (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1 -phosphate synthase);

(WO00/11177; and U.S. Patent 6,225,529). For dicots, seed-preferred promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nucl. See also, WOOO/12733, where seed-preferred promoters from END1 and END2 genes are disclosed.

The term “inducible promoter” refers to promoters that selectively express a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals.

Inducible or regulated promoters include, for example, promoters induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress,

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PCT/US2016/057279 phytohormones, wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners.

An example of a stress-inducible is RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91). One of ordinary skill in the art is familiar with protocols for simulating drought conditions and for evaluating drought tolerance of plants that have been subjected to simulated or naturally-occurring drought conditions. For example, one can simulate drought conditions by giving plants less water than normally required or no water over a period of time, and one can evaluate drought tolerance by looking for differences in physiological and/or physical io condition, including (but not limited to) vigor, growth, size, or root length, or in particular, leaf color or leaf area size. Other techniques for evaluating drought tolerance include measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates. Also, one of ordinary skill in the art is familiar with protocols for simulating stress conditions such as osmotic stress, salt stress and temperature stress and for evaluating stress tolerance of plants that have been subjected to simulated or naturally-occurring stress conditions.

Another example of an inducible promoter useful in plant cells has been described in US patent application, US 2013-0312137A1, published on November 21, 2013, incorporated by reference herein. US patent application US 201320 0312137A1 describes a ZmCASI promoter from a CBSU-Anther_Subtraction library (CAS1) gene encoding a mannitol dehydrogenase from maize, and functional fragments thereof. The ZmCASI promoter (also refered to as “CAS1 promoter”, “mannitol dehydrogenase promoter “, “mdh promoter”) can be induced by a chemical or stress treatment. The chemical can be a safener such as, but not limited to, N-(aminocarbonyl)-2-chlorobenzenesulfonamide (2-CBSU). The stress treatment can be a heat treatment such as, but not limited to, a heat shock treatment (see also US provisional patent application, 62/120421, filed on February 25, 2015, and incorporated by reference herein.

New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) In The Biochemistry of Plants, Vol. 115, Stumpf and Conn, eds (New York, NY: Academic Press), pp. 1-82.

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PCT/US2016/057279 “Translation leader sequence” refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (e.g., Turner and Foster, (1995) Mol Biotechnol 3:225-236).

“3’ non-coding sequences”, “transcription terminator” or “termination sequences” refer to DNA sequences located downstream of a coding sequence and io include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3’ end of the mRNA precursor. The use of different 3’ non-coding sequences is exemplified by Ingelbrecht etal., (1989) Plant Cell 1:67115 680.

“RNA transcript” refers to the product resulting from RNA polymerasecatalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complimentary copy of the DNA sequence, it is referred to as the primary transcript or pre-m RNA. A RNA transcript is referred to as the mature RNA or mRNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript pre mRNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “crDNA” refers to a DNA that is complementary to, and synthesized from, an mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I.

“Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (see, e.g., U.S. Patent No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5’ non-coding sequence, 3’ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or

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PCT/US2016/057279 other RNA that may not be translated but yet has an effect on cellular processes. The terms “complement” and “reverse complement” are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions can be operably linked, either directly or indirectly, 5’ to the target mRNA, or 3’ to the target mRNA, or within the target mRNA, or a first complementary region is 5’ and its complement is 3’ to the target mRNA.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory. Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis of specific DNA segments and consists of a series of repetitive denaturation, annealing, and extension cycles. Typically, a double-stranded DNA is heat denatured, and two primers complementary to the 3’ boundaries of the target segment are annealed to the DNA at low temperature, and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis, or manipulation of isolated segments of nucleic acids by genetic engineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double-stranded DNA. Such elements may be

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PCT/US2016/057279 autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell. “Transformation cassette” refers to a specific vector containing a gene and having elements in addition to the gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a gene and having elements in addition to the gene that allow for expression of that gene in a host.

The terms “recombinant DNA molecule”, “recombinant construct”, “expression construct”, “ construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not all found together in nature. For example, a construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells. The skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression (Jones et al., (1985) EMBO J 4:2411-2418; De Almeida et al., (1989) Mol Gen

Genetics 218:78-86), and thus that multiple events are typically screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished standard molecular biological, biochemical, and other assays including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.

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The term “expression”, as used herein, refers to the production of a functional end-product (e.g., an mRNA, guide RNA, or a protein) in either precursor or mature form.

The term “introducing” includes reference to introducing, providing, contacting 5 a compound, such as but not limited to, a nucleic acid (e.g., expression construct) or peptide, polypeptide or protein into a cell. Introducing includes the direct delivery of polynucleotides (such as RNA, DNA, RNA-DNA hibrids, single or double stranded oligonucleotides, linear or circular polynucleotides) and/or includes the direct delivery of proteins (polypeptides). Introducing includes reference to the incorporation of a nucleic acid or polypeptide into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient introduction of a nucleic acid or protein into the cell. Introducing includes reference to stable or transient transformation methods, transfection, transduction, microinjection, electroporation, viral methods,

Agrobacterium-med\ated transformation, ballistic particle acceleration, whiskers mediated transformation, as well as sexually crossing. Thus, “introducing” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct, guide RNA, guide DNA, template DNA, donor DNA) into a cell, includes “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

A variety of methods are known for introducing, contacting and/or providing a composition into an organisms including stable transformation methods, transient transformation methods, virus-mediated methods, sexual crossing and sexual breeding. Stable transformation indicates that the introduced polynucleotide integrates into the genome of the organism and is capable of being inherited by progeny thereof. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

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Protocols for contacting, providing, introducing polynucleotides and polypeptides into cells or organisms are known and include microinjection (Crossway etal., (1986) Biotechniques 4:320-34 and U.S. Patent No. 6,300,543), meristem transformation (U.S. Patent No. 5,736,369), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6, Agrobacterium-med\ated transformation (U.S. Patent Nos. 5,563,055 and 5,981,840), whiskers mediated transformation (Ainley et al. 2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide (2011), 345-358 Editor(s): Gerhardt, Rosario. Publisher: InTech, Rijeka, Croatia.

io CODEN: 69PQBP; ISBN: 978-953-307-201-2) direct gene transfer (Paszkowski et al., (1984) EMBO J 3:2717-22), and ballistic particle acceleration (U.S. Patent Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes etal., (1995) Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg & Phillips (Springer-Verlag, Berlin); McCabe etal., (1988) Biotechnology 6:923-6; Weissinger etal., (1988) Ann Rev Genef 22:421-77; Sanford et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev Biol 27P:175-82 (soybean); Singh et al., (1998) TheorAppl Genet 96:319-24 (soybean); Datta et al., (1990)

Biotechnology 8:736-40 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci. USA

85:4305-9 (maize); Klein et al., (1988) Biotechnology 6:559-63 (maize); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein etal., (1988) Plant Physiol 91:4404 (maize); Fromm et al., (1990) Biotechnology 8:833-9 (maize); Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Patent No. 5,736,369 (cereals);

Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae)., De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal., (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) TheorAppl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-505 (electroporation); Li et al., (1993) Plant Cell Rep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996) Nat Biotechnol 14:745-50 (maize via Agrobacterium tumefaciens).

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Alternatively, polynucleotides may be introduced into cells or organisms by contacting cells or organisms with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule. In some examples a polypeptide of interest may be initially synthesized as part of a viral polyprotein, which is later processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known, see, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931. Transient transformation methods include, but io are not limited to, the introduction of polypeptides, such as a double-strand break inducing agent, directly into the organism, the introduction of polynucleotides such as DNA and/or RNA polynucleotides, and the introduction of the RNA transcript, such as an mRNA encoding a double-strand break inducing agent, into the organism. Such methods include, for example, microinjection or particle bombardment. See, for example Crossway et al., (1986) Mol Gen Genet 202:17985; Nomura etal., (1986) Plant Sci 44:53-8; Hepler etal., (1994) Proc. Natl. Acad. Sci. USA 91:2176-80; and, Hush etal., (1994) J Cell Sc/107:775-84.

Nucleic acids and proteins can be provided to a cell by any method including methods using molecules to facilitate the uptake of anyone or all components of a guided Cas system (protein and/or nucleic acids), such as cell-penetrating peptides and nanocariers. See also US20110035836 Nanocarier based plant transfection and transduction, and EP 2821486 A1 Method of introducing nucleic acid into plant cells, incorporated herein by reference.

Introducing a guide RNA/Cas endonuclease complex into a cell includes introducing the individual components of said complex either separately or combined into the cell, and either directly (direct delivery as RNA for the guide and protein for the Cas endonuclease) or via recombination constructs expressing the components (guide RNA, Cas endonuclease). Introducing a guide RNA/Cas endonuclease complex into a cell includes introducing the guide RNA/Cas endonuclease complex as a ribonucleotide-protein into the cell. The ribonucleotide-protein can be assembled prior to being introduced into the cell as described herein.

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Plant cells differ from human and animal cells in that plant cells contain a plant cell wall which may act as a barrier to the direct delivery of the RGEN ribonucleoproteins and/or of the direct delivery of the RGEN components.

As described herein, direct delivery of the RGEN ribonucleoproteins into plant 5 cells can be achieved through particle mediated delivery (particle bombardment.

Based on the experiments described herein, a skilled artesian can now envision that any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, electroporation, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, can be successfully used for delivering RGEN ribonucleoproteins into plant cells.

Direct delivery of the RGEN ribonucleoprotein, as described herein, allows for genome editing at a target site in the genome of a cell which can be followed by rapid degradation of the complex, and only a transient presence of the complex in the cell. This transient presence of the RGEN complex may lead to reduced off15 target effects. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can result in constant expression of RGENs from these plasmids which can intensify off target effects (Cradick, T. J. et al (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al (2014) Nat. Biotechnol. 31:822-826.

Direct delivery can be achieved by combining any one component of the RNA guided endonuclease (guide RNA, Cas protein, mRNA encoding the gRNA or Cas endonuclease) or the RGEN complex itself, with a particle delivery matrix comprising a microparticle such as but not limited to of a gold particle, tungsten particle, and silicon carbide whisker particle. Examples of combination methods described herein for combining microparticles to plasmid DNA and DNA of interest can also be used for coating guide RNA molecules, mRNA molecules, Cas proteins and RGEN complexes to the microparticles.

These coated microparticles can be introduced into the cells by any direct method known in the art such as the particle bombardment method described in

Example 8. Microparticles and RGEN components or RGEN complex can be combined (mixed) in any matter to allow for coating of the RGEN components to the mirco particles. For example, RGEN components can be precipitated onto gold

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PCT/US2016/057279 pellets of a diameter ranging from at least 0.1 pm, 0.2 pm, 0.3 pm , 0.4 pm ,0.5 pm,0.6 pm ,0.7 pm ,0.8 pm ,0.9 pm or 1.0 pm in diameter using any suitable buffer (such as but not limiting to a water-soluble cationic lipid such as but not limiting to TranslT-2020 Transfection Reagent (Cat# MIR 5404, Mirus, USA). RGEN component solutions can prepared on ice (or at any temperature suitable to enable mircoparticle bounding) using at least 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg ,0.5 pg ,0.6 pg, 0.7 pg ,0.8 pg ,0.9 pg, 1.0 pg, 2.0 pg , 3.0 pg , 4.0 pg ,5.0 pg ,6.0 pg ,7.0 pg ,8.0 pg 9.0 pg or 10 pg of RNA (guided RNA or mRNA) or Cas endonuclease protein. To the pre-mixed RGEN components of RGEN complexes, at least 1 pi to 20 pi of io prepared mircoparticles can be added and mixed carefully.

In one embodiment of the disclosure, the method comprises a method of delivering a guide RNA /Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotideprotein and matrix to bind and form a ribonucleotide-protein - matrix complex; and, introducing said ribonucleotide-protein - matrix complex into said cell. The particle delivery matrix can comprise microparticles combined with a cationic lipid.

The term “cationic lipid” includes reference to a water soluble cationic lipid, such as but not limiting to TranslT-2020, or a cationic lipid solution such as but not limiting to a cationic lipid solution comprising N,N,N',N'-tetramethyl-N, N'-bis(2hydroxylethyl)-2,3-di( oleoyloxy )-1 ,4-butanediammonium iodide, and L-dioleoyl phosphatidylethanolamine (DOPE).(see also US2007/0178593, published on August 2, 2007, incorporated herein by reference),

5. The method of claim.

The particle delivery matrix can comprise microparticles selected from the group consisting of gold particles, tungsten particles, and silicon carbide whisker particles.

The particle delivery matrix can further comprise a compound selected from 30 the group consisting of Tfx-10™, Tfx-20™, Tfx-50™, Lipofectin™, Lipofectamine™,

Cellfectin™, Effectene™, Cytofectin GSV™, Perfect Lipids™, DOTAP™, DMRIEC™, FuGENE-6™, Superfect™, Polyfeet™, polyethyleneimine, chitosan, protamine

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Cl, histone H1, histone CENH3, poly-L lysine, and DMSA.(US2007/0178593, published on August 2, 2007, incorporated herein by reference)

RGEN components can also be combined prior to be coated on microparticles by combining least 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg ,0.5 pg ,0.6 pg ,0.7 pg ,0.8 pg ,0.9 pg, 1.0 pg, 2.0 pg , 3.0 pg , 4.0 pg ,5.0 pg ,6.0 pg ,7.0 pg ,8.0 pg 9.0 pg or 10 pg of guide RNA with at least 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg ,0.5 pg ,0.6 pg ,0.7 pg ,0.8 pg ,0.9 pg, 1.0 pg, 2.0 pg, 3.0 pg , 4.0 pg ,5.0 pg ,6.0 pg ,7.0 pg ,8.0 pg 9.0 pg or 10 pg of Cas endonuclease in a solution suitable to allow for complex formation (such as but not limiting to a Cas9 buffer (NEB)), at any temperature to allow for io complex formation such as a temperature ranging from 1°C, 2°C , 3°C, 4°C ,5°C, 6°C ,7°C ,8°C, 9°C ,10°C, 11°C, 12°C , 13°C , 14°C, 15°C, 16°C, 17°C ,18°C,

19°C, 20°C, 21 °C, 22°C, 23.0°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33.0°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C and 40°C.

In one embodiment of the disclosure, the method comprises a method of delivering guide RNA /Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said guide RNA and said Cas endonuclease protein to form a complex inside said cell.

In one embodiment of the disclosure, the method comprises a method of delivering guide RNA /Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one mRNA encoding a Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said mRNA to translate said Cas endonuclease protein and form a complex with said guide RNA

In one embodiment of the disclosure, the method comprises a method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotideprotein and matrix to bind and form a ribonucleotide-protein - matrix complex; and, introducing said ribonucleotide-protein - matrix complex together with at least one a

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PCT/US2016/057279 polynucleotide template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii).

In one embodiment of the disclosure, the method comprises a method of delivering a guide RNA/Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotideprotein and matrix to bind and form a ribonucleotide-protein - matrix complex; and, introducing said ribonucleotide-protein - matrix complex together with a donor DNA into said cell,, wherein said donor DNA comprises at least one polynucleotide of interest.

Suitable conditions for growing cells are well known in the art and the skilled artesian can use any growing condition based on the type of cell (such as conditions suitable for plant cells). As described in Example 8, plant embryos or cells can be incubated in any plant maintenance medium known in the art (such as, but not limiting to 560P, Example 8) for 12 to 48 hours at temperatures ranging from 26°C to 37°C, and then placed at 26°C. After 5 to 7 days the embryos/cells are transferred to any selection medium known in the art (such as, but not limiting to 560R, Example 8), and subcultured thereafter.

RGEN components (including guide RNA, Cas endonuclease protein) can be combined to form a ribonucleotide-protein complex (RNP) prior to be coated on (combined with) microparticles by combining least 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg ,0.6 pg ,0.7 pg ,0.8 pg ,0.9 pg, 1.0 pg, 2.0 pg, 3.0 pg, 4.0 pg, 5.0 pg, 6.0 pg, 7.0 pg, 8.0 pg, 9.0 pg or 10 pg of guide RNA with at least 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1.0 pg, 2.0 pg, 3.0 pg, 4.0 pg, 5.0 pg, 6.0 pg, 7.0 pg, 8.0 pg, 9.0 pg or 10 pg of Cas endonuclease in a solution suitable to allow for complex formation (such as but not limiting to a Cas9 buffer (NEB)), at any temperature to allow for complex formation such as a temperature ranging from 1°C,

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2°C , 3°C, 4°C ,5°0, 6°C_>7°C_>8°C, 9°C ,10°C, 11°C, 12°C, 13°C, 14°0, 15°0, 16°0, 17°C J8°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°0, 25°0, 26°0, 27°Ο, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°0, 35°0, 36°0, 37°Ο, 38°C, 39°C and 40°C.

“Mature” protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed). “Precursor” protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acid fragment into a io genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or other DNAcontaining organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms.

The commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. Gene stacking can be accomplished by many means including but not limited to co-transformation, retransformation, and crossing lines with different genes of interest.

Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, and plant cells as well as plants and seeds produced by the methods described herein. Plant cells include cells selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cells.

The term “plant” includes reference to whole plants, plant organs, plant tissues, seeds, and plant cells, and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant parts include differentiated and undifferentiated tissues

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PCT/US2016/057279 including, but not limited to roots, stems, shoots, leaves, pollens, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos, and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture. The term plant organ refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term genome refers to the entire complement of genetic material (genes and noncoding sequences) that is present in each cell of an organism, or virus or organelle; and/or a complete set of chromosomes inherited as a (haploid) unit from one parent. “Progeny” comprises any subsequent generation of a plant.

A transgenic plant includes, for example, a plant which comprises within its genome a heterologous polynucleotide introduced by a transformation step. The heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant

DNA construct. A transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant. A heterologous polynucleotide can include a sequence that originates from a foreign species, or, if from the same species, can be substantially modified from its native form. Transgenic can include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The alterations of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods, by the genome editing procedure described herein that does not result in an insertion of a foreign polynucleotide, or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be regarded as transgenic.

In certain embodiments of the disclosure, a fertile plant is a plant that produces viable male and female gametes and is self-fertile. Such a self-fertile plant can produce a progeny plant without the contribution from any other plant of a gamete and the genetic material contained therein. Other embodiments of the

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PCT/US2016/057279 disclosure can involve the use of a plant that is not self-fertile because the plant does not produce male gametes, or female gametes, or both, that are viable or otherwise capable of fertilization. As used herein, a male sterile plant is a plant that does not produce male gametes that are viable or otherwise capable of fertilization. As used herein, a female sterile plant is a plant that does not produce female gametes that are viable or otherwise capable of fertilization. It is recognized that male-sterile and female-sterile plants can be female-fertile and male- fertile, respectively. It is further recognized that a male fertile (but female sterile) plant can produce viable progeny when crossed with a female fertile plant and that a female fertile (but male sterile) plant can produce viable progeny when crossed with a male fertile plant.

Non-conventional yeast herein refers to any yeast that is not a Saccharomyces (e.g., S. cerevisiae) or Schizosaccharomyces yeast species. Nonconventional yeast are described in Non-Conventional Yeasts in Genetics,

Biochemistry and Biotechnology: Practical Protocols (K. Wolf, K.D. Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003), which is incorporated herein by reference. Non-conventional yeast in certain embodiments may additionally (or alternatively) be yeast that favor non-homologous end-joining (NHEJ) DNA repair processes over repair processes mediated by homologous recombination (HR).

Definition of a non-conventional yeast along these lines - preference of NHEJ over HR - is further disclosed by Chen et al. (PLoS ONE 8:e57952), which is incorporated herein by reference. Preferred non-conventional yeast herein are those of the genus Yarrowia (e.g., Yarrowia lipolytica). The term “yeast” herein refers to fungal species that predominantly exist in unicellular form. Yeast can alternative be referred to as “yeast cells” herein (see also US provisional application 62/036,652, filed on August 13, 2014, which is incorporated by reference herein).

A centimorgan (cM) or map unit is the distance between two linked genes, markers, target sites, loci, or any pair thereof, wherein 1% of the products of meiosis are recombinant. Thus, a centimorgan is equivalent to a distance equal to a 1% average recombination frequency between the two linked genes, markers, target sites, loci, or any pair thereof.

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The present disclosure finds use in the breeding of plants comprising one or more introduced traits. Most commonly, transgenic traits are randomly inserted throughout the plant genome as a consequence of transformation systems based on Agrobacterium, biolistics, or other commonly used procedures. More recently, gene targeting protocols have been developed that enable directed transgene insertion. One important technology, site-specific integration (SSI) enables the targeting of a transgene to the same chromosomal location as a previously inserted transgene. Custom-designed meganucleases and custom-designed zinc finger meganucleases allow researchers to design nucleases to target specific chromosomal locations, and these reagents allow the targeting of transgenes at the chromosomal site cleaved by these nucleases.

The currently used systems for precision genetic engineering of eukaryotic genomes, e.g. plant genomes, rely upon homing endonucleases, meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), which require de novo protein engineering for every new target locus. The highly specific, RNA-directed DNA nuclease, guide RNA/ Cas9 endonuclease system described herein, is more easily customizable and therefore more useful when modification of many different target sequences is the goal.

The guide RNA/Cas system described herein is especially useful for genome engineering, especially plant genome engineering, in circumstances where nuclease off-target cutting can be toxic to the targeted cells. In one embodiment of the guide RNA/Cas system described herein, an expression-optimized Cas9 gene, is stably integrated into the target genome, e.g. plant genome. Expression of the Cas9 gene is under control of a promoter, e.g. plant promoter, which can be a constitutive promoter, tissue-specific promoter or inducible promoter, e.g. temperature-inducible, stress-inducible, developmental stage inducible, or chemically inducible promoter.

In the absence of the guide RNA or crRNA, the Cas9 protein is not able to cut DNA and therefore its presence in the plant cell should have little or no consequence. Hence a key advantage of the guide RNA/Cas system described herein is the ability to create and maintain a cell line or transgenic organism capable of efficient expression of the Cas9 protein with little or no consequence to cell viability. In order to induce cutting at desired genomic sites to achieve targeted genetic modifications,

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PCT/US2016/057279 guide RNAs or crRNAs can be introduced by a variety of methods into cells containing the stably-integrated and expressed cas9 gene. For example, guide RNAs or crRNAs can be chemically or enzymatically synthesized, and introduced into the Cas9 expressing cells via direct delivery methods such a particle bombardment or electroporation. Alternatively, genes capable of efficiently expressing guide RNAs or crRNAs in the target cells can be synthesized chemically, enzymatically or in a biological system, and these genes can be introduced into the Cas9 expressing cells via direct delivery methods such a particle bombardment, electroporation or biological delivery methods such as Agrobacterium mediated

DNA delivery.

A guide RNA/Cas system mediating gene targeting can be used in methods for directing transgene insertion and I or for producing complex transgenic trait loci comprising multiple transgenes in a fashion similar as disclosed in

WO2013/0198888 (published August 1, 2013) where instead of using a double 15 strand break inducing agent to introduce a gene of interest, a guide RNA/Cas system as disclosed herein is used. A complex trait locus includes a genomic locus that has multiple transgenes genetically linked to each other. By inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5 , 1.0, 2, or even 5 centimorgans (cM) from each other, the transgenes can be bred as a single genetic locus (see, for example, U.S. patent application 13/427,138) or PCT application PCT/US2012/030061. After selecting a plant comprising a transgene, plants containing (at least) one transgenes can be crossed to form an F1 that contains both transgenes. In progeny from these F1 (F2 or BC1) 1/500 progeny would have the two different transgenes recombined onto the same chromosome. The complex locus can then be bred as single genetic locus with both transgene traits. This process can be repeated to stack as many traits as desired.

Chromosomal intervals that correlate with a phenotype or trait of interest can be identified. A variety of methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to the gene controlling the trait of interest. In other words, the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the

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PCT/US2016/057279 interval) can be used as a marker for northern leaf blight resistance. In one embodiment, the chromosomal interval comprises at least one QTL, and furthermore, may indeed comprise more than one QTL. Close proximity of multiple QTLs in the same interval may obfuscate the correlation of a particular marker with a particular QTL, as one marker may demonstrate linkage to more than one QTL. Conversely, e.g., if two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear if each of those markers identifies the same QTL or two different QTL. The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question. An “allele of a QTL” can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers. A haplotype can be defined by the unique fingerprint of alleles at each marker within the specified window.

A variety of methods are available to identify those cells having an altered genome at or near a target site without using a screenable marker phenotype. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof.

Proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of the protein(s) can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci.

USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.

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Guidance regarding amino acid substitutions not likely to affect biological activity of the protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by routine screening assays. Assays for double-strandbreak-inducing activity are known and generally measure the overall activity and io specificity of the agent on DNA substrates containing target sites.

The term “dicot” refers to the subclass of angiosperm plants also knows as “dicotyledoneae” and includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

The term “crossed” or “cross” or “crossing” in the context of this disclosure means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule (or microspores and megaspores) are from the same plant or genetically identical plants).

The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a transgene, a modified (mutated or edited) native allele, or a selected allele of a marker or QTL.

Standard DNA isolation, purification, molecular cloning, vector construction, and verification/characterization methods are well established, see, for example

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Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY). Vectors and constructs include circular plasmids, and linear polynucleotides, comprising a polynucleotide of interest and optionally other components including linkers, adapters, regulatory or analysis. In some examples a recognition site and/or target site can be contained within an intron, coding sequence, 5' UTRs, 3' UTRs, and/or regulatory regions.

The present disclosure further provides expression constructs for expressing in a plant, plant cell, or plant part a guide RNA/Cas system that is capable of binding to and creating a double strand break in a target site. In one embodiment, the io expression constructs of the disclosure comprise a promoter operably linked to a nucleotide sequence encoding a Cas gene and a promoter operably linked to a guide RNA of the present disclosure. The promoter is capable of driving expression of an operably linked nucleotide sequence in a plant cell.

Any plant can be used, including monocot and dicot plants. Examples of monocot plants that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), wheat (Triticum aestivum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palm, ornamentals, turfgrasses, and other grasses. Examples of dicot plants that can be used include, but are not limited to, soybean (Glycine max), canola (Brassica napus and B. campestris), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum) etc.

The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “pL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “pM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “pmole” mean micromole(s), “g” means gram(s), “pg” means microgram(s), “ng” means

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PCT/US2016/057279 nanogram(s), “U” means unit(s), “bp” means base pair(s) and “kb” means kilobase(s).

Non-limiting examples of compositions and methods disclosed herein are as follows:

1. A method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable io of encoding said functional gene product.

2. The method of embodiment 1, wherein said disrupted gene comprises a base pair deletion of the 4^th nucleotide upstream (5’) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional.

3. The method of embodiment 2, wherein the base pair deletion is the first nucleotide of a codon sequence.

4. The method of embodiment 2, wherein the base pair deletion is the second nucleotide of a codon sequence.

5. The method of embodiment 2, wherein the base pair deletion is the third nucleotide of a codon sequence.

6. The method of embodiment 1, wherein the restoration is accomplished by NonHomologous-End -Joining (NHEJ) resulting in the insertion of a single base at the double strand break site.

7. The method of embodiment 1, wherein the restoration is accomplished by the insertion of a single base at the double strand break site without the use of Homologous Recombination or homology-directed repair.

8. A method for modifying a nucleotide sequence in the genome of a cell, the method comprising:

introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a

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PCT/US2016/057279 first complex capable of introducing a double strand in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide RNA and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable protein .

9. The method of embodiment 8, wherein the modification is selected from the group consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, or a substitution of at least one nucleotide in said target site.

10. The method of embodiment 8, further comprising introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence.

11. The method of embodiment 8, wherein the at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii).

12. The method of embodiment 8, further comprising introducing a donor DNA into the cell of (a) wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site.

13. The method of embodiment 8, wherein the cell is selected from the group consisting of a human, non-human, animal, archaea, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell.

14. The method of embodiment 13, wherein the plant cell is selected from the group consisting of a monocot and dicot cell.

15. The method of embodiment 14, wherein the plant cell is selected from the group consisting of a maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cell.

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16. The method of embodiment 13, further comprising producing a plant or progeny plant from said plant cell.

17. A plant or progeny plant produced by the method of embodiment 16, wherein said plant or progeny plant is void of any one guide RNA and Cas endonucleases.

18. The method of embodiment 8, wherein the disrupted selectable marker gene is a disrupted ALS-resistance gene.

19. A method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising:

a) introducing into at least one cell at least one guide RNA and at least one Cas io endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence;

b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and,

c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.

20. A method for editing a nucleotide sequence in the genome of a plant without the use of a polynucleotide modification template or donor DNA, the method comprising:

a) introducing into at least one plant cell at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence;

b) selecting a plant cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited;

c) regenerating a plant from the plant cell of (b);

d) introducing into a cell from the plant of (c) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas

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PCT/US2016/057279 endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and inserting a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template; and,

e) optimally, selecting a cell comprising the nucleotide insertion of (d).

21. The method of any one of embodiments 1, 8, 18-20, wherein the guide RNA and Cas endonuclease protein forming the guide RNA /Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

22. The method of any one of embodiments 1, 8, 18-20, wherein the guide RNA io /Cas endonuclease complex is assembled in vitro prior to being introduced intyo the plant cell, and introduced into the cell as a ribonucleotide-protein complex.

23. The method of any one of embodiments 1, 8, 18-20 wherein components of the guide RNA/Cas endonuclease complex are introduced as mRNAencoding the Cas endonuclease protein and as RNA comprising the guide RNA.

24. The method of any one of embodiments 1, 8, 18-19 wherein components of the guide RNA/Cas endonuclease complex are introduced as recombinant DNA molecules encoding the guide molecule and the Cas endonuclease protein.

25. The method of any one of embodiments 1,8,18-19 wherein the guide RNA /Cas endonuclease complex is assembled inside the cell.

26. A method of delivering a guide RNA /Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotideprotein and matrix to bind and form a ribonucleotide-protein - matrix complex; and, introducing said ribonucleotide-protein - matrix complex into said cell.

27. A method of delivering guide RNA /Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least one Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said guide RNA and said Cas endonuclease protein to form a complex inside said cell.

28. A method of delivering guide RNA /Cas endonuclease components into a cell, the method comprising introducing at least one guide RNA molecule and at least

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PCT/US2016/057279 one mRNA encoding a Cas endonuclease protein into a cell, and growing said cell under suitable conditions to allow said mRNA to translate said Cas endonuclease protein and form a complex with said guide RNA.

29. The method of embodiments 26-28, further comprising introducing a polynucleotide template, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) io an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii).

30. The method of embodiments 26-28, further comprising introducing a donor DNA, wherein said donor DNA comprises at least one polynucleotide of interest.

31. The method of embodiments 26-28, wherein said guide RNA /Cas endonuclease complex introduces a double strand break at a target site in the genome of said cell.

32. The method of embodiments 1-31, wherein said Cas endonuclease is selected from the group consisting of a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-H, Cas 5, Cas7, Cas8, Casio, or complexes of these.

33. The method of embodiments 26-28, wherein the introducing is via a delivery system selected from the group consisting of particle mediated delivery, whisker mediated delivery, cell-penetrating peptide mediated delivery, electroporation, PEPmediated transfection and nanoparticle mediated delivery.

34. The method of embodiment 29, wherein the polynucleotide modification template is a single stranded or double stranded molecule.

35. The method of embodiment 30, wherein the donor DNA is a single stranded or double stranded molecule.

36. The method of embodiments 26-28, wherein the cell is a plant cell that comprises pre-integrated developmental genes capable of stimulating cell development.

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37. The method of embodiments 26-28, wherein the cell is selected from the group consisting of a human, non-human, animal, archaea, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell.

38. The method of embodiment 37, wherein the plant cell is selected from the group consisting of a monocot and dicot cell.

39. The method of embodiment 38, wherein the plant cell is selected from the group consisting of a maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cell.

io 40. A plant produced from the plant cell of embodiment 36, wherein said plant comprises said at least one nucleotide modification in the genome of said plant cell and wherein said plant does not comprise said guide RNA/Cas endonuclease complex or any component thereof.

41. A plant produced from the plant cell of embodiment 37, wherein said plant comprises at least one polynucleotide of interest integrated into its genome.

42. The method of embodiment 26, wherein said particle delivery matrix comprises a microparticle.

43. The method of embodiment 42, wherein said microparticle is selected from the group consisting of a gold particle, a tungsten particle, and a silicon carbide whisker particle.

44. The method of embodiment 26, wherein the particle delivery matrix comprises microparticles combined with a cationic lipid.

45. The method of embodiment 44, wherein the cationic lipid is a water soluble cationic lipid.

46. The method of embodiment 45, wherein the water soluble cationic lipid is

TranslT-2020.

47. A method for producing a sulfonylurea resistant plant comprising a modified target site, the method comprising: a) introducing to a plant cell comprising a disrupted sulfonylurea resistant (ALS) gene, a first guide RNA, a Cas9 endonuclease, at least a second guide RNA, wherein said first guide RNA and Cas9 endonuclease can form a first complex capable of introducing a double strand break immediately downstream (3’) of a second nucleotide of a codon sequence located in

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PCT/US2016/057279 said disrupted sulfonylurea resistant (ALS) gene, wherein said second guide RNA and Cas9 endonuclease can form a second complex capable of introducing a double strand break at said target site; and, b) obtaining a sulfonylurea resistant plant from said plant cell, wherein said sulfonylurea resistant plant comprises a modification at said target, wherein said modification is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

48. A method for restoring function to a non-functional gene product in the genome io of a cell, the method comprising introducing a guide polynucleotide/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product.

49. The method of embodiment 46, wherein said disrupted gene comprises a base pair deletion of the 4^th nucleotide upstream (5’) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional.

48. A method for modifying a nucleotide sequence in the genome of a cell, the method comprising:

introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide polynucleotide, a Cas endonuclease, and at least a second guide polynucleotide, wherein said first guide polynucleotide and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored without the use of a polynucleotide modification template to a nondisrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide polynucleotide and Cas endonuclease can form a second complex that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and,

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PCT/US2016/057279 selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein.

50. A method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising:

a) introducing into at least one cell at least one guide polynucleotide and at least one Cas endonuclease, wherein said guide polynucleotide and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence;

b) selecting a cell from (a) comprising at least one single nucleotide deletion in io said nucleotide sequence, wherein said nucleotide deletion is located at a position to be edited; and,

c) introducing into a cell of (b) at least one guide polynucleotide and at least one Cas endonuclease, wherein said guide polynucleotide and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide deletion of (b) without the use of a polynucleotide modification template.

51. A method of delivering a guide polynucleotide /Cas endonuclease complex into a cell, the method comprising combining at least one guide polynucleotide molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotide-protein and matrix to bind and form a ribonucleotide-protein - matrix complex; and, introducing said ribonucleotide-protein - matrix complex into said cell.

EXAMPLES

In the following Examples, unless otherwise stated, parts and percentages are by weight and degrees are Celsius. It should be understood that these Examples, while indicating embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Such modifications are also intended to fall within the scope of the appended claims.

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EXAMPLE 1

Modifying target DNA sequences in the genome of a plant cell by delivering

Cas9 endonuclease and guide RNA expression cassettes The Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) (SEQ ID

NO: 1) was maize codon optimized using standard techniques known in the art and the potato ST-LS1 intron (SEQ ID NO: 2) was introduced in order to eliminate its expression in E.coli and Agrobacterium. To facilitate nuclear localization of the Cas9 protein in maize cells, Simian virus 40 (SV40) monopartite amino terminal nuclear localization signal (MAPKKKRKV, SEQ ID NO: 3) and Agrobacterium io tumefaciens bipartite VirD2 T-DNA border endonuclease carboxyl terminal nuclear localization signal (KRPRDRHDGELGGRKRAR, SEQ ID NO: 4) were incorporated at the amino and carboxyl-termini of the Cas9 open reading frame, respectively.

The maize optimized Cas9 gene was operably linked to a maize constitutive promoter (Ubiquitin) by standard molecular biology techniques. Transcription is terminated by the addition of the 3’ sequences from the potato proteinase inhibitor II gene (Pinll) to generate UBI:Cas9:Pinll vector. The sequence of the Ubiquitin driven maize optimized Cas9 expression cassette is shown in SEQ ID NO: 5.

Single guide RNAs (gRNAs) were designed using the methods described by Mali et al., 2013 (Science 339:823-26). A maize U6 polymerase III promoter and terminator were isolated and used to direct initiation and termination of gRNAs, respectively. Two Bbs\ restriction endonuclease sites were introduced in an inverted tandem orientation with cleavage orientated in an outward direction as described in Cong et al., 2013 (Science 339:819-23) to facilitate the rapid introduction of maize genomic DNA target sequences into the gRNA expression constructs. Only target sequences starting with a G nucleotide were used to promote favorable polymerase III expression of the gRNA. The gRNA expression cassettes were subcloned into Bluescript SK vector (SEQ ID NO: 6).

To test whether the maize optimized Cas9-gRNA complex could recognize, cleave, and facilitate targeted mutations in maize chromosomal DNA through non30 homologous end joining (NHEJ) repair pathway, 5 maize loci (three different genomic sequences in each locus) were targeted for cleavage (see Table 2) and examined by amplicon deep sequencing for the presence of mutations.

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Table 2. Maize genomic sites targeted by the Cas9-gRNA system

Locus	Location	Target Site Designation	Maize Genomic Target Site Sequence	PAM Sequence	SEQ ID NO:
MS26	Chr. 1: 51.81cM	MS26Cas-1	GTACTCCATCCGCCCCATCGAGTA	GGG	7
MS26Cas-2	GCACGTACGTCACCATCCCGC	CGG	8
MS26Cas-3	GACGTACGTGCCCTACTCGAT	GGG	9
LIG	Chr. 2: 28.45cM	LIGCas-1	GTACCGTACGTGCCCCGGCGG	AGG	10
LIGCas-2	GGAATTGTACCGTACGTGCCC	CGG	11
LIGCas-3	GCGTACGCGTACGTGTG	AGG	12
MS45	Chr. 9: 119.15cM	MS45Cas-1	GCTGGCCGAGGTCGACTAC	CGG	13
MS45Cas-2	GGCCGAGGTCGACTACCGGC	CGG	14
MS45Cas-3	GGCGCGAGCTCGTGCTTCAC	CGG	15
ALS1 and ALS2	1 - Chr. 4: 107.73cM 2 - Chr. 5: 115.49cM	ALSCas-1	GGTGCCAATCATGCGTCG	CGG	16
ALSCas-2	GGTCGCCATCACGGGAC	AGG	17
ALSCas-3	GTCGCGGCACCTGTCCCGTGA	TGG	18

MS26=Male Sterility Gene 26, LIG=LigulelessA Gene Promoter, MS45=Male

Sterility Gene 45, ALS1=Acetolactate Synthase Gene 1 (Chr.4), ALS1=Acetolactate Synthase Gene 2 (Chr.5).

The maize optimized Cas9 endonuclease and gRNA expression cassettes containing the specific maize variable targeting domains were co-delivered to 60-90 Hi-Il immature maize embryos by particle bombardment (see Example 8) with io selectable and visible marker (UBI:MoPAT:DsRED fusion) and developmental genes ZmODP-2 (BBM) and ZmWUS2 (WUS) (see Example 9). Hi-Il maize embryos transformed with only the Cas9 or gRNA expression cassette served as

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PCT/US2016/057279 negative controls. After 7 days, 20-30 most uniformly transformed embryos from each treatment (based on transient expression of the DsRED fluorescent protein) were pooled and total genomic DNA was extracted. The region surrounding the intended target site was PCR amplified with Phusion® High Fidelity PCR Master Mix (New England Biolabs, M0531L) adding sequences necessary for amplicon-specific barcodes and lllumnia sequencing using “tailed” primers through two rounds of PCR. The primers used in the primary PCR reaction are shown in Table 3.

Table 3. PCR primer sequences

Target Site	Primer	Primary PCR Primer Sequence	SEQ ID NO:
MS26Cas-1	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TAGGACCGGAAGCTCGCCGCGT	19
MS26Cas-1	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TTCCTGGAGGACGACGTGCTG	20
MS26Cas-2	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TAAGGTCCTGGAGGACGACGTGCTG	21
MS26Cas-2	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TCCGGAAGCTCGCCGCGT	22
MS26Cas-3	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TTCCTCCGGAAGCTCGCCGCGT	23
MS26Cas-3	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TTCCTGGAGGACGACGTGCTG	20
LIGCas-1	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TAGGACTGTAACGATTTACGCACCTGCTG	24
LIGCas-1	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCAAATGAGTAGCAGCGCACGTAT	25
LIGCas-2	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TTCCTCTGTAACGATTTACGCACCTGCTG	26

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LIGCas-2	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCAAATGAGTAGCAGCGCACGTAT	25
LIGCas-3	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TAAGGCGCAAATGAGTAGCAGCGCAC	27
LIGCas-3	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TCACCTGCTGGGAATTGTACCGTA	28
MS45Cas-1	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TAGGAGGACCCGTTCGGCCTCAGT	29
MS45Cas-1	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCCGGCTGGCATTGTCTCTG	30
MS45Cas-2	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TTCCTGGACCCGTTCGGCCTCAGT	31
MS45Cas-2	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCCGGCTGGCATTGTCTCTG	30
MS45Cas-3	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TGAAGGGACCCGTTCGGCCTCAGT	32
MS45Cas-3	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCCGGCTGGCATTGTCTCTG	30
ALSCas-1	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TAAGGCGACGATGGGCGTCTCCTG	33
ALSCas-1	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCGTCTGCATCGCCACCTC	34
ALSCas-2	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TTTCCCGACGATGGGCGTCTCCTG	35
ALSCas-2	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCGTCTGCATCGCCACCTC	34

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ALSCas-3	Forward	CTACACTCTTTCCCTACACGACGCTCTTCCGATC TGGAACGACGATGGGCGTCTCCTG	36
ALSCas-3	Reverse	CAAGCAGAAGACGGCATACGAGCTCTTCCGATC TGCGTCTGCATCGCCACCTC	34

Primers used in the secondary PCR reaction were AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG (forward, SEQ ID NO: 37) and CAAGCAGAAGACGGCATA (reverse, SEQ ID NO: 38).

The resulting PCR amplifications were purified with a Qiagen PCR purification spin column, concentration measured with a Hoechst dye-based fluorometric assay, combined in an equimolar ratio, and single read 100 nucleotidelength deep sequencing was performed on lllumina’s MiSeq Personal Sequencer with a 30-40% (v/v) spike of PhiX control v3 (lllumina, FC-110-3001) to off-set io sequence bias. Only those reads with a >1 nucleotide indel arising within the 10 nucleotide window centered over the expected site of cleavage and not found in a similar level in the negative control were classified as mutations. Mutant reads with the same mutation were counted and collapsed into a single group and the top 10 most prevalent mutations were visually confirmed as arising within the expected site of cleavage. The total numbers of mutations were then used to calculate the percentage of mutant reads based on the total number of reads of an appropriate length containing a perfect match to the barcode and forward primer.

The mutation frequencies revealed by amplicon deep sequencing for the Cas9-gRNA system targeting all 15 sites are shown in Table 4.

Table 4. Percent of mutant reads at 5 target loci (15 target sites)

Target	DSB Reagents	Total Number of Reads	Number of Mutant Target Gene Reads	Percentage of Mutant Reads in Target Gene
LIG (Chr. 2)	LIG-CR1 gRNA+Cas9	716,854	33,050	4.61%
LIG-CR2 gRNA+Cas9	711,047	16,675	2.35%

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	LIG-CR3 gRNA+Cas9	713,183	27,959	3.92%
MS26 (Chr. 1)	MS26-CR1 gRNA+Cas9	575,671	10,073	1.75%
MS26-CR2 gRNA+Cas9	543,856	16,930	3.11%
MS26-CR3 gRNA+Cas9	538,141	13,879	2.58%
MS45 (Chr. 9)	MS45-CR1 gRNA+Cas9	812,644	3,795	0.47%
MS45-CR2 gRNA+Cas9	785,183	14,704	1.87%
MS45-CR3 gRNA+Cas9	728,023	9,203	1.26%
ALS1 (Chr. 4) and ALS2 (Chr. 5)	ALS-CR1 gRNA+Cas9	434,452	9,669	2.23%
ALS-CR2 gRNA+Cas9	472,351	6,352	1.35%
ALS-CR3 gRNA+Cas9	497,786	8,535	1.72%
Controls	Cas9 only	640,063	1	0.00%
LIG-CR1 gRNA only	646,774	1	0.00%

Further analysis demonstrated, that the most common type of mutations promoted by Cas9-gRNA system was single nucleotide insertions (for example, see Figure 1, SEQ ID NOs: 49-58). Similar results were observed for the majority of gRNAs tested (Table 5).

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Table 5. Frequency of a single nucleotide insertions and deletions in 15 target sites promoted by the Cas9-gRNA system

Target Locus	DSB Reagents	% Single nt Insertion of Total Number of Mutant Reads	% Single nt Deletion of Total Number of Mutant Reads
LIG	LIG-CR1 gRNA+Cas9	86%	5%
LIG-CR2 gRNA+Cas9	49%	25%
LIG-CR3 gRNA+Cas9	62%	20%
MS26	MS26-CR1 gRNA+Cas9	46%	16%
MS26-CR2 gRNA+Cas9	78%	8%
MS26-CR3 gRNA+Cas9	45%	18%
MS45	MS45-CR1 gRNA+Cas9	45%	17%
MS45-CR2 gRNA+Cas9	41%	23%
MS45-CR3 gRNA+Cas9	20%	24%
ALS1 (Chr. 4) and ALS2 (Chr. 5)	ALS-CR1 gRNA+Cas9	22%	76%
ALS-CR2 gRNA+Cas9	60%	27%
ALS-CR3 gRNA+Cas9	84%	12%

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This example demonstrates that RNA guided Cas9 generates double strand breaks resulting in high frequency of mutations. Analysis of mutations in multiple target sites showed that although various size deletions and/or insertions were observed, a single nucleotide insertion and a single nucleotide deletion were the most prevalent types of mutations generated by the Cas9-gRNA technology for the majority of the target sites tested in maize.

EXAMPLE 2

Edited Acetolactate Synthase Gene Confers Resistance to Chlorsulfuron

This example demonstrates that specific change(s) introduced into the io nucleotide sequence of the native maize acetolactate synthase (ALS) gene result in resistance to sulfonylurea class herbicides, specifically, chlorsulfuron.

There are two ALS genes in maze, ALS1 (SEQ ID NO: 39) and ALS2 (SEQ ID NO: 40), located on chromosomes 4 and 5, respectively, with 94% sequence identity at the DNA level.

The ALS protein contains N-terminal transit and the mature protein is formed following transport into the chloroplast and subsequent cleavage of the transit peptide. The mature protein starts at residue S41, resulting in a mature protein of 598 amino acids with a predicted molecular weight of 65 kDa (SEQ ID NO: 41).

Modification of a nucleotide sequence of either ALS1 or ALS2 resulting in a single amino acid residue (P165A or P165S, boxed in grey) change in comparison to the endogenous maize acetolactate synthase protein provides resistance to herbicides in maize.

As acetolactate synthase is a critical enzyme for cell function in plants, simultaneous bi-allelic knockouts of ALS1 and ALS2 genes would not be expected to survive.

Therefore, based on polymorphism between ALS1 and ALS2 nucleotide sequences, ALS2-specific ALSCas-4 target site was identified and tested. ALSCas-1 guide RNA expressing construct targeting both ALS1 and ALS2 genes was used as control. Table 6 presents information about ALSCas-1 and ALSCas-4 target sites.

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Table 6. ALSCas-1 and ALSCas-4 (ALS2-specific) target sites

Loci	Location	Target Site Designatio n	Maize Genomic Target Site Sequence	PAM Sequence	SEQ ID NO:
ALS1 and ALS2	Chr. 4: 107.73cM and Chr. 5: 115.49cM	ALSCas-1	GGTGCCAATCATGCGTCG	CGG	16
ALSCas-4	GCTGCTCGATTCCGTCCCCA	TGG	42

Underlined nucleotides in the ALSCas-4 target site and PAM are different in the ALS1 gene.

Mutation frequencies at the ALSCas-1 and ALSCas-4 were determined by 5 amplicon deep sequencing as described in Example 1 and shown in Table 7.

Table 7. Frequencies of mutations at ALSCas-1 and ALSCas-4 target sites recovered by amplicon deep sequencing.

Target Site	Total Reads	Mutant reads (ALS1)	Mutant reads (ALS2)
ALSCas-1	204,230	2704 (1.3%)	5072 (2.5%)
ALSCas-4	120,766	40 (0.03%)	3294 (2.7%)

These results demonstrated that ALSCas-4 gRNA/Cas9 system mutated the io ALS2 gene with approximately 90 times higher efficiency than the ALS1 gene.

Therefore, the ALSCas-4 target site and the corresponding ALS-CR4 gRNA were selected for the ALS gene editing experiment.

To generate ALS2 edited alleles, a 794 bp polynucleotide modification template comprising a fragment of homology (SEQ ID NO: 43) was cloned into a plasmid vector and two 127 nt single-stranded polynucleotide modification templates (also referred to as DNA oligos, Oligol, SEQ ID NO: 44, and Oligo2, SEQ ID NO:

45) were tested as polynucleotide modification templates (Figure 2). The 794 bp fragment had the same sequence modifications as Oligol. The polynucleotide modification templates (repair templates) contained several nucleotide changes in comparison to the native sequence. Single-stranded Oligol and the 794 bp repair templates included a single nucleotide change which would direct editing of DNA sequences corresponding to the proline at amino acid position 165 to a serine (P165S), as well as three additional changes within the ALS-CR4 target site and

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PAM sequence. Modification of the PAM sequence within the repair template altered the methionine codon (AUG) to isoleucine (AUU), which naturally occurs in the ALS1 gene. A second 127 nt single-stranded oligo repair template (Oligo2) was also tested which preserved the methionine at position 157 but contained three additional single nucleotide changes in the sequence which would influence base pairing with the ALS-CR4 gRNA (Figure 2).

Approximately 1,000 immature embryos per treatment were bombarded with the two oligo or single plasmid repair templates, Cas9, ALS-CR4 gRNA, and MoPAT-DsRED in DNA expression cassettes and placed on media to select for io bialaphos resistance conferred by PAT. Five weeks post-transformation, two hundred (per treatment) randomly selected independent young callus sectors growing on selective media were separated from the embryos and transferred to fresh bialaphos plates. The remaining embryos (> 800 per treatment) with developing callus events were transferred to the plates containing 100 ppm of chlorosulfuron as direct selection for an edited ALS2 gene. A month later, a total of 384 randomly picked callus sectors growing on bialaphos (approximately 130 events for each repair template) and 7 callus sectors that continued growing on media with chlorsulfuron were analyzed by PCR amplification and sequencing. Edited ALS2 alleles were detected in nine callus sectors: two derived from the callus sectors growing on bialaphos and generated using the 794 bp repair DNA template, and the remaining 7 derived from chlorosulfuron resistant callus sectors edited using the 127 nt single-stranded oligos, three by Oligol and four by Oligo2. The second ALS2 allele in these callus sectors was mutated as a result of NHEJ repair. Analysis of the ALS1 gene revealed only wild-type sequence confirming high specificity of the ALS25 CR4 gRNA.

Plants were regenerated from 7 out of 9 callus sectors containing edited ALS2 alleles for additional molecular analysis and progeny testing. DNA sequence analysis of ALS2 alleles confirmed the presence of the P165S modifications (ALS2P165S) as well as the other nucleotide changes associated with the respective repair templates. T1 and T2 progeny of two TO plants generated from different callus events (794 bp repair DNA and Oligo2) were analyzed to evaluate the inheritance of the edited ALS2 alleles. Progeny plants derived from crosses using pollen from wild

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PCT/US2016/057279 type Hi-Il plants were analyzed by sequencing and demonstrated sexual transmission of the edited alleles observed in the parent plant with expected 1:1 segregation ratio (57:56 and 47:49, respectively). To test whether the edited ALS sequence confers herbicide resistance, selected four-week old segregating T1 plants with edited and wild-type ALS2 alleles were sprayed with four different concentrations of chlorsulfuron (50, 100 (1x), 200, and 400 mg/liter). Three weeks after treatment, plants with an edited allele showed normal phenotype (Figure 3 left), while plants with only wild-type alleles demonstrated strong signs of senescence (Figure 3-right).

io In addition to resistance to sulfonylurea class herbicides (specifically, chlorsulfuron), ALS genes can be modified to confer resistance to other classes of AHAS inhibitors including triazolopy-rimidines, pyrimidinylthio-benzoates, and Imidazolinone herbicides (Tan S, Evans RR, Dahmer ML, Singh BK, Shaner DL (2005) Imidazolinone-tolerant crops: history, current status and future. Pest

Management Science 61: 246-257). Thus, modifications to ALS genes should not be limited to changes describe herein and conferring chlorsulfuron resistance.

These experiments demonstrate that Cas9-gRNA can stimulate HDRdependent targeted sequence modifications in maize resulting in plants with an edited endogenous gene which properly transmits to subsequent generations. The data also indicate that a single edited ALS2 allele under endogenous promoter provides herbicide resistance in maize.

EXAMPLE 3

ALS2 as Endogenous Selectable Marker Gene

This example demonstrates how specifically edited ALS2 gene can be used 25 to generate a selectable marker in a cell replacing delivery of exogenous marker genes currently used in plant transformation.

Due to the relatively low frequencies of plant transformation (transgenic event recovery), selectable marker genes providing resistance to various herbicides are routinely co-delivered with trait genes. To confer resistance, these selectable marker genes need to be stably integrated into the plant genome and have to be excised or bred out in consecutive generations. Some native plant genes can be specifically modified (edited) to confer resistance to herbicides. As described in Example 2, the

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ALS2 gene with a single amino acid change provides resistance to chlorsulfuron. Therefore, it may be anticipated that gene mutagenesis, gene editing or co-delivery of a trait gene and coincident ALS2 gene editing can be used without an exogenously supplied marker gene. Giving high mutation frequency as the result of

NHEJ repair of DSB generated by Cas9-gRNA system (Example 1), this approach might be useful for gene mutagenesis. In this case, the frequency of mutated events would be anticipated to be dependent on HDR-mediated ALS gene editing. With respect to gene editing, it is likely that the combination of two low frequency HDRdependent genome editing processes (one for ALS gene repair for selection and io another for endogenous gene editing or trait gene integration) in plant cells would make the approach using coincident ALS2 gene editing rather impractical.

The following example describes a method that allows overcoming this low efficiency (and impracticality) and improving the likelihood of selecting for plant cells resistant to selective agents. The method does not rely on HDR-dependent gene editing but rather relies on the restoration of the gene function by targeted mutagenesis through NHEJ DNA repair, which is more common (than HDR) in plant somatic cells. As described in Example 2, there are two ALS genes in maize, ALS1 and ALS2, located on chromosomes 4 and 5, respectively. Specific editing of either one of the two ALS genes will confer herbicide resistance. These genes play an essential role in plant metabolism, consequently, targeting and mutating both of them at the same time leads to the cell death. Therefore, in this example, modifications only involve the ALS2 gene by using an ALS2-specific gRNA that does not target the ALS1 gene, hence ALS1 remains wild type. Specifically, two modifications are introduced into the ALS2 gene; first, specific nucleotide(s) change, for example, C to T at the nucleotide position 493 (Figure 2, oligol) or C to T and C to G at the nucleotide positions 493 and 495, respectively (Figure 2, oligo2) to convert Proline to Serine at amino acid position 165 (named ALS2-P165S) conferring resistance to chlorsulfuron (see Example 2 for details). Second, removal of a single nucleotide, for example a G at the nucleotide position 165 (Figure 4A-4B) resulting in the translational frameshift (Figure 4B) and, hence, loss of ALS2mediated chlorsulfuron resistance (named ALS2-P165S-CCA). While many designs are anticipated, for the highest frequency of ALS2 gene repair, the single nucleotide

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PCT/US2016/057279 position (example shown in Figure 4A-4C) should preferably be : i) the 4^th nucleotide upstream (5’) from the PAM sequence in a gRNA/Cas endonuclease target site, and ii) the 3^rd nucleotide in the codon (Figure 4A). This 3^rd position is flexible for most amino acids and can be occupied by any of the four nucleotides in

8 out of 20 amino acids (Table 8). Given such flexibility, a higher frequency of proper repair is anticipated at the 3^rd position, when compared to the 1^st or 2^nd position.

Table 8. Genetic code.

Amino Acid	Codons	Compressed Codons
Alanine / Ala	GCU, GCC, GCA, GCG	GCN
Arginine / Arg	CGU, CGC, CGA, CGG, AGA, AGG	CGN, MGR
Glycine / Gly	GGU, GGC, GGA, GGG	GGN
Leucine / Leu	CUU, CUC, CUA, CUG, UUA, UUG	CUN, YUR
Proline / Pro	CCU, CCC, CCA, CCG	CCN
Serine / Ser	UCU, UCC, UCA, UCG, AGU, AGC	UCN, AGY
Threonine / Thr	ACU, ACC, ACA, ACG	ACN
Valine / Val	GUU, GUC, GUA, GUG	GUN
Isoleucine / lie	AUU, AUC, AUA	AUH
Asparagine / Asn	AAU, AAC	AAY
Aspartic Acid / Asp	GAU, GAC	GAY
Cysteine / Cys	UGU, UGC	UGY
Glutamine / Gin	CAA, CAG	CAR
Glutamic Acid / Glu	GAA, GAG	GAR
Histidine / His	CAU, CAC	CAY
Lysine / Lys	AAA, AAG	AAR
Phenylalanine / Phe	UUU, UUC	UUY
Tyrosine / Tyr	UAU, UAC	UAY
Methionine / Met	AUG	-
Tryptophan / Trp	UGG	-

Four different target sites satisfying the above criteria and the corresponding gRNAs were selected. Besides the above stated preferred single nucleotide position, the corresponding gRNA should promote high frequency of mutations with high percentage of mutations representing a single nucleotide insertion at the cleavage site. Only one of the four target sites tested satisfied all the described

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PCT/US2016/057279 preferences and, therefore, suitable for this experiment, was identified (referred to as ALSCas-7; Table 9).

Table 9. ALS2-specific ALSCas-7 target site and ALS-CR7- gRNA evaluation by amplicon deep sequencing.

Target	Target Site Sequence	% Of Mutant Reads	% of Mutant Reads with 1 bp Insertion	% of Mutant Reads with 1 bp Deletion	SEQ ID NO:
ALSCas-4 (control)	GCTGCTCGATTCCGTCCCCA	2.73%	-	-	42
ALSCas-7	GCTCCCCCGGCCACCCCGCTC	2.99%	2.23% (75%)	0.14% (5%)	79

Then, the ALS2-P165S gene conferring resistance to chlorsulfuron was further modified (resulting in a disrupted gene): the proline codon encoded by CCG (underlined in Table 9) in the ALSCas-7 target site was altered by removal of the G io nucleotide at the wobble position (3^rd nucleotide position in the codon) resulting in the translational frameshift and a disrupted gene (referred to as ALS2-P165S-CCA; Figure 4 A-4B). As demonstrated in Example 1, repair of DSBs generated by Cas9gRNA system in maize, and repaired through NHEJ, often results in a single nucleotide insertion at the cleavage site. Therefore, the function of the ALS2-P165S15 CCA gene, and consecutively, cell resistance to chlorsulfuron, can be restored by generating a double strand break (DSB) at the modified ALSCas-7 site, referred to as ALSCas-7-1 (GCTCCCCCGGCCACCCCCTC; SEQ ID NO: 80) and its repair through NHEJ (see Figure 4B-4C).

Based on this disclosure, one can envision simultaneous delivery of two or more gRNAs when one gRNA targets and activates the disrupted ALS2-P165SCCA gene through NHEJ (thus conferring herbicide resistance) and the other gRNA(s) promote DSB(s) at a site(s) different than ALS2 and facilitate desired genome modifications, for example, targeted mutagenesis, deletion, gene editing, or site-specific trait gene insertions. This approach can allow for completely transient

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PCT/US2016/057279 targeted genome modifications as all other necessary components (Cas9, gRNAs) can be delivered in a form of protein and/or in vitro transcribed RNA molecules.

To test this approach, maize plants (Hi-Il genotype) with specifically modified the ALS2 gene described above were generated. First, ALS2 sequence was modified at the amino acid position 165 to confer resistance to chlorsulfuron as described in Example 2. Immature embryos from plants homozygous for the edit were then bombarded with Cas9 and ALS-CR7 gRNA targeting ALSCas-7 target site, selectable marker (UBI:MoPAT-DSRED fusion) and cell developmental enhancing genes (for details, see Examples 8 and 9). Regenerants from bialaphos io resistant callus sectors were analyzed by sequencing. Several TO plants with a single nucleotide deletion (a G in the nucleotide position 165) were identified (Figure 4A-4B). This deletion resulted in the translational frameshift (Figure 4B) and, hence, loss of ALS2-P165S-mediated chlorsulfuron resistance. Plants homozygous for both edits (ALS2-P165S-CCA) were regenerated, confirmed by sequencing and tested for the loss of herbicide resistance by spraying with chlorsulfuron.

Embryos from homozygous plants with specifically modified endogenous

ALS2 gene (ALS2-P165S-CCA) were used in a prove-of-concept experiment. In order to demonstrate that an edited disrupted ALS2-P165S-CCA gene can be repaired as described above (so that it encodes a functional protein) and work as selectable marker, DNA vectors encoding for Cas9, gRNA targeting the ALSCas-7-1 site (refed as ALS-CR7-1), cell development enhancing genes (ZmODP2 and ZmWUS), and MS45-CR2 gRNA were co-delivered into maize (Hi-Il) immature embryo cells. One week after bombardment, embryos were transferred to the media with 100ppm chlorsulfuron for selection. Approximately 30% of embryos (84 out of

290) developed herbicide resistant callus events, which were analyzed by sequencing. The vast majority of the events (79 events) demonstrated a single nucleotide insertion at the expected ALSCas-7-1 DSB site (Figure 4C) and complete restoration of the ALS2-P165S gene. Four events had no insertion but either 2 or 5 bp deletions putting the gene back in frame, and a single event that seemed to be an escape. Fifty out of 83 events (60%) also demonstrated mutations at the MS45Cas-2 target site.

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This example demonstrates prove-of-concept and utility of a specifically modified, inactive ALS2 as endogenous selectable marker gene by the use of a guided Cas endonuclease system. Based on the results described herein, one skilled in the art can use and expand the described approach to any similarly modified endogenous or pre-integrated exogenous gene(s) replacing co-delivery of a selectable marker gene currently used in plant genome editing experiments.

EXAMPLE 4

Alternative Designs to Restore Function to a Non-functional Protein Encoded by a

Disrupted Gene.

io In the previous example, sequence alterations were incorporated within the coding region or ALS2-P165S. It is anticipated that others sequence changes which create a disrupted gene (that does not encode a functional protein) can also be designed to be used as re-activation sequences. This example describes generation of a re-activation sequence that does not depend upon the restoration of a codon within a coding sequence, but rather the elimination of a start codon which is upstream and out-of-frame of the primary translational start codon of ALS2-P165S.

According to a scanning model of eukaryotic translation initiation the first AUG codon relative to the 5’ cap of an mRNAs is used to initiate protein synthesis (Kozak M. 1989. The scanning model for translation: an update. The Journal of Cell

Biology 108: 229-241). Thus, if an AUG codon within the non-coding leader of the

RNA transcript is upstream and out-of-frame of the primary start codon, protein synthesis of the polypeptide encoded by the mRNA is abolished. To take advantage of this rule and apply to a strategy of reactivation of gene expression or function, an endogenous ALS2-P165S allele can be generated to contain an upstream out-of25 frame translational start codon (Figure 5A-5B). This allele contains a Cas9 PAM recognition site and an ALS-CRX targeting spacer. Cutting by Cas9 between nucleotides 3 and 4, located 5’ of the PAM site in this example can promote nucleotide deletion(s) or addition(s) which can result in the loss of the ATG codon. Loss of this upstream out-of-frame ATG by any combination of deletion or addition due to NHEJ repair can result in translation initiation at the primary ALS2-P165S start codon conferring herbicide resistance.

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It is anticipated that the re-activation strategy using an upstream out-of-frame ATG not be limited to the design in Figure 5A-5B. PAM and targeting spacer can also be placed at various positions relative to the upstream out-of-frame ATG, as long as targeted cutting by Cas9 results in the loss of this start codon. For example, the PAM can be present on the antisense strand, 5’ of the start codon. Other designs can be contemplated; the out-of-frame ATG start codon can also be placed at different positions within the 5’ leader sequence. The PAM sequence can be recognized by other Cas9 proteins like Streptococcus pyogenes which recognize nGG PAMs or non-nGG PAM sequences for example Streptococcus thermophiles io CR1 (PAM sequence recognition nnAGAAn) and others having PAM sequences. The utility of other Cas9 proteins would satisfy the re-activation design of this example as well as Example 3 described above.

Other designs for gene activation are anticipated. As mentioned earlier, in addition to chlorosulfuron resistance, modifications to the ALS gene which confer resistance to other herbicides can be used for reactivation. Furthermore, the phosphomannose isomerase gene (PMI), bialaphos resistance gene (BAR), phosphinothricin acetyltransferase (PAT), hygromycin resistance gene (NPTII), selectable marker genes, fluorescent marker genes (such as but not limiting to RFP, red fluorescent protein, CFP, GFP, green fluorescent protein) and glyphosate resistance genes can be modified to be introduced into plant cells as inactive forms and used as targets for re-activation by guide RNA introduction and repair by NHEJ as described in Example 3. It would be also anticipated that having multiple inactive genes can serve as targets. For example, guide RNA multiplexing has been demonstrated to simultaneously modify multiple genes in a single experiment, thus targeted reactivation of chlorsulfuron and bialaphos resistance, but not limited to these genes, can be an additionally designed for this approach. As described above, coincident with restoration of gene function by NHEJ, modification of other targets would be accomplished simultaneously by the addition of other guidepolynucleotides.

In addition, it would be anticipated that similar approach can be applied to any native or introduced gene sequence and used as an efficient gene switch mechanism.

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EXAMPLE 5

Specific gene editing without introducing polynucleotide modification templates for homology directed repair.

Example 2 described sequence alterations within the coding region of the

ALS2 gene (P165S) using specifically designed polynucleotide modification templates (repair templates). The example below describes a different approach to generating an edited gene of interest that does not depend upon the HDR mechanism, but rather on NHEJ.

As described in Example 1, two of the most prevalent types of mutations io facilitated by NHEJ repair of DSBs generated by Cas9 nuclease are 1 bp insertion and 1 bp deletion (Table 4). Based on these observations described herein, methods were developed for gene editing that can be accomplished in two consecutive steps or into a single step, as described below.

The first step includes targeting a gene or polynucleotide of interest, containing a target site that is recognized by a Cas endonuclease, using the RNA guided Cas nuclease system described herein, resulting in the generation of a cell or an organism with a specific nucleotide deletion due to NHEJ repair of the cleaved DNA (illustrated in Figure 6A). The second step requires re-targeting the mutated site and selecting events with insertion of a desired nucleotide (without the use of a polynucleotide modification template (repair template), hence, specifically changing the corresponding amino acid and the gene function. In general, the idea is illustrated in Figure 6A-6C. This method can also be used to edit non-coding DNA fragments.

Alternatively, both steps can be combined into a single step. Two different gRNAs, one recognizing the original target site and the second one the same site but with a 1 bp deletion can be used. In this case, one can envision a consecutive cutting and repair of the endogenous site resulting in a 1 bp deletion followed by cutting of the altered site and its repair with a 1 bp insertion. Then, an event with an insertion of a desired nucleotide can be selected. In the case of editing coding DNA sequences, this process accomplishes two goals - restoring the translational reading frame and replacement of an amino acid of interest. It would be anticipated that combinations of different endonuclease could be used in this two-step or one111

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PCT/US2016/057279 step system. For example, the introduction of a first double strand break leading to a single base deletion in a polynucleotide of interest can be accomplished by a first endonuclease, whereas the introduction of a second double strand break leading to a single base insertion (and editing of the polynucleotide of interest) can be accomplished by a second endonuclease that is different from the first endonuclease. The differences between the endonucleases may include, but are not limited to, different PAM recognition sequences, different target recognition sequences, different cleavage activity (blunt-end, 5’ or 3’ overhang, single strand, double strand), different DNA or amino acid sequences, originating from different io organism, or any one combination thereof.

The ability to edit a specific nucleotide in a genome of interest may depend on the endonuclease system of choice and its ability to recognize and cut a particular target site. The discovery of novel guided endonucleases (See for example US patent application 62/162377 filed May 15 2015), and/or modifications of guided endonucleases with various PAM sequences, will further increase the density of target sites that can be recognized and/or cleaved by these endonuclease ultimately resulting in the ability to target any given nucleotide position in the genome using the methods described herein.

EXAMPLE 6

Maize Lines with Stably Integrated Cas9 Endonuclease

This example describes generation and validation of maize lines with stably integrated Cas9 expression cassette.

Two Agrobacterium vectors (Figure 7, containing maize-codon optimized

Cas9 under the transcriptional control of a constitutive (maize UBI, SEQ ID NO: 46) or a temperature regulated (maize MDH, SEQ ID NO: 47) promoter were introduced into Hi-Il embryo cells to establish lines containing pre-integrated genomic copies of Cas9 endonuclease. These vectors also contained an embryo-preferred END2 promoter regulating the expression of a blue-fluorescence gene (AmCYAN) as a visible marker and an interrupted copy of the DsRED gene transcriptionally regulated by a maize Histone 2B promoter. Part of the DsRED sequence was duplicated in a direct orientation (369 bp fragment) and consisted of two fragments of the DsRED (RF-FP) gene which were separated by a 347 bp spacer that could be

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PCT/US2016/057279 targeted by gRNAs. DSBs within the spacer region promote intramolecular recombination restoring function to the disrupted DsRED gene which results in red fluorescing cells. Maize plants with single-copy T-DNA inserts containing either UBI:Cas9 or MDH:Cas9 were used as a source of immature embryos. Blue5 fluorescing embryos containing pre-integrated Cas9 were excised and incubated at 28°C (UBI:Cas9) or at 37°C (MDH:Cas9) for 24 hours. Post-bombardment, embryos with MDH:Cas9 were incubated at 37°C for 24 hours and then moved to 28°C. In contrast to control (no gRNA), UBI:Cas9 and MDH:Cas9 containing embryos bombarded with two DNA-expressed gRNAs that targeted sequences io within the 347 bp spacer, readily produced red fluorescing foci.

These results demonstrate that described above maize lines poses single copies of functional Cas9 endonuclease.

EXAMPLE 7

Transient gRNA Delivery into Embryo Cells with Pre-Integrated Cas9 Generates

Mutations in Maize

This Example demonstrates that delivery of gRNA in a form of in vitro transcribed RNA molecules into maize immature embryo cells with pre-integrated Cas 9 generates mutations at targeted sites.

Maize plants described in Example 6 containing either UBI:Cas9 or

MDH:Cas9 were used as a source of immature embryos for delivery of gRNAs as in vitro transcribed RNA or as DNA expression cassettes as control. To measure mutation frequencies at the LIG and MS26 endogenous target sites, LIG-CR3 and MS26-CR2 gRNAs as RNA molecules (100 ng/shot) or as DNA vectors (25 ng/shot) were delivered into UBI:Cas9 and MDH:Cas9 containing embryo cells with temperature treatments described in Example 6. In these experiments, embryos were harvested two days post-bombardment and analyzed by amplicon deep sequencing. Similar frequencies were detected for gRNAs delivered as DNA vectors and as RNA molecules, particularly in the case of Cas9 regulated by the Ubiquitin promoter (Table 10).

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Table 10. Percentage of mutant reads at maize LIG and MS26 target sites produced by transient gRNA delivery into embryos with pre-integrated Cas9 under constitutive (UBI) or regulated (MDH) promoters

Target Site	Embryos	Transformation	Percentage of Mutant Reads (2 days post-bombardment)
LIG	UBI:Cas9	gRNA (DNA)	1.22%
gRNA(RNA)	1.86%
MDH:Cas9 event 1	gRNA (DNA)	0.25%
gRNA(RNA)	0.12%
MDH:Cas9 event 2	gRNA (DNA)	0.57%
gRNA(RNA)	0.26%
MDH:Cas9 event 3	gRNA (DNA)	0.46%
gRNA(RNA)	0.35%
MS26	MDH:Cas9 event 2	gRNA (DNA)	0.58%
gRNA(RNA)	0.17%

Together, these data demonstrate that delivery of gRNA in the form of RNA directly into maize cells containing pre-integrated Cas9 is a viable alternative to DNA delivery for the generation of mutations in plant cells.

EXAMPLE 8

Transformation of Maize Immature Embryos io Transformation can be accomplished by various methods known to be effective in plants, including particle-mediated delivery, Agrobacterium-med\ated transformation, PEG-mediated delivery, and electroporation.

a. Particle-mediated delivery

Transformation of maize immature embryos using particle delivery is performed as follows. Media recipes follow below.

The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5%

Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are isolated and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the

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2.5-cm target zone in preparation for bombardment. Alternatively, isolated embryos are placed on 560L (Initiation medium) and placed in the dark at temperatures ranging from 26°C to 37°C for 8 to 24 hours prior to placing on 560Y for 4 hours at 26°C prior to bombardment as described above.

Plasmids containing the double strand brake inducing agent and template or donor DNA are constructed using standard molecular biology techniques and cobombarded with plasmids containing the developmental genes ODP2 (AP2 domain transcription factor ODP2 (Ovule development protein 2); US20090328252 A1) and WUSCHEL (US2011/0167516).

io The plasmids and DNA of interest are precipitated onto 0.6 pm (average diameter) gold pellets using a water-soluble cationic lipid TranslT-2020 Transfection Reagent (Cat# MIR 5404, Mirus, USA) as follows. DNA or DNA and RNA solution is prepared on ice using a total of 1 pg of DNA and/or RNA constructs (10 shots). To the pre-mixed DNA, 20 pi of prepared gold particles (15 mg/ml) and 1 pi TransYT15 2020 are added and mixed carefully. Gold particles are pelleted in a microfuge at

10,000 rpm for 1 min and supernatant is removed. 105 pi of 100% EtOH is added and the particles are resuspended by brief sonication. Then, 10 pi is spotted onto the center of each macrocarrier and allowed to dry before bombardment. The sample plates are bombarded using Biorad Helium Gun (shelf #3) at 425 PSI.

Following bombardment, the embryos are incubated on 560P (maintenance medium) for 12 to 48 hours at temperatures ranging from 26C to 37C, and then placed at 26C. After 5 to 7 days the embryos are transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks at 26C. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to a lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to a 2.5 pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to Classic 600 pots (1.6 gallon) and grown to maturity. Plants are

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Initiation medium (560L) comprises 4.0 g/l N6 basal salts (SIGMA C-1416),

1.0 ml/l Eriksson’s Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCI, 20.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-l H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-l H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Maintenance medium (560P) comprises 4.0 g/l N6 basal salts (SIGMA C10 1416), 1.0 ml/l Eriksson’s Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCI,

30.0 g/l sucrose, 2.0 mg/l 2,4-D, and 0.69 g/l L-proline (brought to volume with D-l H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-l H2O); and 0.85 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA ΟΙ 416), 1.0 ml/l Eriksson’s Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCI, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-l H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-l H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).

Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416),

1.0 ml/l Eriksson’s Vitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCI, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-l H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with

D-l H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117 074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished

D-l H2O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myoinositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-l H2O after adjusting to pH 5.6); 3.0 g/l Gelrite

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Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine

HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-l H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-l H2O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-l H2O), sterilized and cooled to 60°C.

b. Agrobacterium-mediated transformation io Agrobacterium-med\ated transformation was performed essentially as described in Djukanovic et al. (2006) Plant Biotech J 4:345-57. Briefly, 10-12 day old immature embryos (0.8 -2.5 mm in size) were dissected from sterilized kernels and placed into liquid medium (4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/L thiamine HCI, 1.5 mg/L 2, 4-D,

0.690 g/L L-proline, 68.5 g/L sucrose, 36.0 g/L glucose, pH 5.2). After embryo collection, the medium was replaced with 1 ml Agrobacterium at a concentration of 0.35-0.45 OD550. Maize embryos were incubated with Agrobacterium for 5 min at room temperature, then the mixture was poured onto a media plate containing 4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E20 1511), 1.0 mg/L thiamine HCI, 1.5 mg/L 2, 4-D, 0.690 g/L L-proline, 30.0 g/L sucrose, 0.85 mg/L silver nitrate, 0.1 nM acetosyringone, and 3.0 g/L Gelrite, pH 5.8. Embryos were incubated axis down, in the dark for 3 days at 20°C, then incubated 4 days in the dark at 28°C, then transferred onto new media plates containing 4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/L thiamine HCI, 1.5 mg/L 2, 4-D, 0.69 g/L L-proline, 30.0 g/L sucrose, 0.5 g/L MES buffer, 0.85 mg/L silver nitrate, 3.0 mg/L Bialaphos, 100 mg/L carbenicillin, and 6.0 g/L agar, pH 5.8. Embryos were subcultured every three weeks until transgenic events were identified. Somatic embryogenesis was induced by transferring a small amount of tissue onto regeneration medium (4.3 g/L MS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/L myo-inositol, 0.1 μΜ ABA, 1 mg/L IAA, 0.5 mg/L zeatin, 60.0 g/L sucrose, 1.5 mg/L Bialaphos, 100 mg/L carbenicillin, 3.0 g/L Gelrite, pH 5.6) and incubation in the dark for two weeks at

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28°C. All material with visible shoots and roots were transferred onto media containing 4.3 g/L MS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution,

100 mg/L myo-inositol, 40.0 g/L sucrose, 1.5 g/L Gelrite, pH 5.6, and incubated under artificial light at 28°C. One week later, plantlets were moved into glass tubes containing the same medium and grown until they were sampled and/or transplanted into soil.

EXAMPLE 9

Transient Expression of ZmODP-2 and ZmWUS Enhances Transformation

Parameters of the transformation protocol can be modified to ensure that the io BBM activity is transient. One such method involves precipitating the BBMcontaining plasmid in a manner that allows for transcription and expression, but precludes subsequent release of the DNA, for example, by using the chemical PEI.

In one example, the BBM plasmid is precipitated onto gold particles with PEI, while the transgenic expression cassette (UBI:MoPAT-GFPm:Pinll; MoPAT is the maize optimized PAT gene) to be integrated is precipitated onto gold particles using the standard calcium chloride method.

Briefly, gold particles were coated with PEI as follows. First, the gold particles were washed. Thirty-five mg of gold particles, 1.0 in average diameter (A.S.I. #162-0010), were weighed out in a microcentrifuge tube, and 1.2 ml absolute

EtOH was added and vortexed for one minute. The tube was incubated for 15 minutes at room temperature and then centrifuged at high speed using a microfuge for 15 minutes at 4oC. The supernatant was discarded and a fresh 1.2 ml aliquot of ethanol (EtOH) was added, vortexed for one minute, centrifuged for one minute, and the supernatant again discarded (this is repeated twice). A fresh 1.2 ml aliquot of

EtOH was added, and this suspension (gold particles in EtOH) was stored at -20oC for weeks. To coat particles with polyethylimine (PEI; Sigma #P3143), 250 pi of the washed gold particle/EtOH mix was centrifuged and the EtOH discarded. The particles were washed once in 100 pi ddH2O to remove residual ethanol, 250 μΙ of 0.25 mM PEI was added, followed by a pulse-sonication to suspend the particles and then the tube was plunged into a dry ice/EtOH bath to flash-freeze the suspension, which was then lyophilized overnight. At this point, dry, coated particles could be stored at -80oC for at least 3 weeks. Before use, the particles

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PCT/US2016/057279 were rinsed 3 times with 250 μΙ aliquots of 2.5 mM HEPES buffer, pH 7.1, with 1x pulse-sonication, and then a quick vortex before each centrifugation. The particles were then suspended in a final volume of 250 pi HEPES buffer. A 25 μΙ aliquot of the particles was added to fresh tubes before attaching DNA. To attach uncoated

DNA, the particles were pulse-sonicated, then 1 pg of DNA (in 5 pi water) was added, followed by mixing by pipetting up and down a few times with a Pipetteman and incubated for 10 minutes. The particles were spun briefly (i.e. 10 seconds), the supernatant removed, and 60 pi EtOH added. The particles with PEl-precipitated DNA-1 were washed twice in 60 pi of EtOH. The particles were centrifuged, the io supernatant discarded, and the particles were resuspended in 45 pi water. To attach the second DNA (DNA-2), precipitation using 7ra/7slT-2020 was used. The 45 pi of particles/DNA-1 suspension was briefly sonicated, and then 5 pi of 100 ng/μΙ of DNA-2 and 1 pi of 7ra/7slT-2020 were added. The solution was placed on a rotary shaker for 10 minutes, centrifuged at 10,000g for 1 minute. The supernatant was removed, and the particles resuspended in 60 pi of EtOH. The solution was spotted onto macrocarriers and the gold particles onto which DNA-1 and DNA-2 had been sequentially attached were delivered into scutellar cells of 10 DAP Hi-Il immature embryos using a standard protocol for the PDS-1000. For this experiment, the DNA-1 plasmid contained a UBI:RFP:Pinll expression cassette, and

DNA-2 contained a UBI:CFP:Pinll expression cassette. Two days after bombardment, transient expression of both the CFP and RFP fluorescent markers was observed as numerous red & blue cells on the surface of the immature embryo. The embryos were then placed on non-selective culture medium and allowed to grow for 3 weeks before scoring for stable colonies. After this 3-week period, 10 multicellular, stably-expressing blue colonies were observed, in comparison to only one red colony. This demonstrated that PEI-precipitation could be used to effectively introduce DNA for transient expression while dramatically reducing integration of the PEI-introduced DNA and thus reducing the recovery of RFPexpressing transgenic events. In this manner, PEI-precipitation can be used to deliver transient expression of BBM and/or WUS2.

For example, the particles are first coated with UBI:BBM:Pinll using PEI, then coated with UBI:MoPAT-YFP using Trans\T-2020, and then bombarded into

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PCT/US2016/057279 scutellar cells on the surface of immature embryos. PEI-mediated precipitation results in a high frequency of transiently expressing cells on the surface of the immature embryo and extremely low frequencies of recovery of stable transformants (relative to the TranslT-2020 method). Thus, it is expected that the PEI-precipitated

BBM cassette expresses transiently and stimulates a burst of embryogenic growth on the bombarded surface of the tissue (i.e. the scutellar surface), but this plasmid will not integrate. The MoPAT-GFP plasmid released from the Ca++/gold particles is expected to integrate and express the selectable marker at a frequency that results in substantially improved recovery of transgenic events. As a control treatment, PEI-precipitated particles containing a UBI:GUS:Pinll (instead of BBM) are mixed with the MoPAT-GFP/Ca++ particles. Immature embryos from both treatments are moved onto culture medium containing 3mg/l bialaphos. After 6-8 weeks, it is expected that GFP+, bialaphos-resistant calli will be observed in the PEI/BBM treatment at a much higher frequency relative to the control treatment (PEI/GUS).

As an alternative method, the BBM plasmid is precipitated onto gold particles with PEI, and then introduced into scutellar cells on the surface of immature embryos, and subsequent transient expression of the BBM gene elicits a rapid proliferation of embryogenic growth. During this period of induced growth, the explants are treated with Agrobacterium using standard methods for maize (see Example 1), with T-DNA delivery into the cell introducing a transgenic expression cassette such as UBI:MoPAT-GFPm:Pinll. After co-cultivation, explants are allowed to recover on normal culture medium, and then are moved onto culture medium containing 3 mg/l bialaphos. After 6-8 weeks, it is expected that GFP+, bialaphos25 resistant calli will be observed in the PEI/BBM treatment at a much higher frequency relative to the control treatment (PEI/GUS).

It may be desirable to “kick start” callus growth by transiently expressing the BBM and/or WUS2 polynucleotide products. This can be done by delivering BBM and WUS2 5'-capped polyadenylated RNA, expression cassettes containing BBM and WUS2 DNA, or BBM and/or WUS2 proteins. All of these molecules can be delivered using a biolistics particle gun. For example 5'-capped polyadenylated BBM and/or WUS2 RNA can easily be made in vitro using Ambion’s mMessage

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PCT/US2016/057279 mMachine kit. RNA is co-delivered along with DNA containing a polynucleotide of interest and a marker used for selection/screening such as UBI:MoPAT-GFPm:Pinll. It is expected that the cells receiving the RNA will immediately begin dividing more rapidly and a large portion of these will have integrated the agronomic gene. These events can further be validated as being transgenic clonal colonies because they will also express the PAT~GFP fusion protein (and thus will display green fluorescence under appropriate illumination). Plants regenerated from these embryos can then be screened for the presence of the polynucleotide of interest.

EXAMPLE 10 io Direct Delivery of gRNA and Cas9 as a guide RNA/Cas endonuclease

Ribonucleotide-protein Complex (RGEN) into Embryo Cells Generates Mutations in

Maize

This example demonstrates that direct delivery of Cas9 in the form of protein and gRNA in the form of in vitro transcribed or chemically synthesized RNA molecules, into maize immature embryo cells generates mutations at the corresponding targeted sites.

To generate gRNA in the form of RNA molecules, the maize-optimized U6 polymerase III gRNA expression cassettes were amplified by PCR using a 5’ oligonucleotide primer that also contained the sequence of the T7 polymerase promoter and transcriptional initiation signal just 5’ of the spacer to gene. T7 in vitro transcription was carried out with the AmpliScribe T7-Flash Kit (Epicentre) according to the manufacturer’s recommendations, and products were purified using NucAway Spin Columns (Invitrogen; Life Technologies Inc) followed by ethanol precipitation.

To generate a guide RNA/Cas9 endonuclease protein complex (RGEN) (also referred to as a guide RNA/Cas9 endonuclease ribonucleotide-protein ), 7 pg of Cas9 (Streptococcus pyogenes Cas9) protein and 3 pg of gRNA molecules (1:2 molar ratio) were mixed in 1x Cas9 buffer (NEB) in a total volume of 20 pi and incubated at room temperature for 15 minutes. Together with the RGEN, plasmids containing Ubiquitin promoter regulated selectable and visible markers (MoPAT30 DsRed fusion), Ubiquitin promoter regulated mays Ovule development protein 2, ZmODP2 (see US20090328252, published December 31,2009) and maize IN2 promoter (Hershey et al. 1991, Plant Mol. Biol 17:679-690) regulated WUSCHEL,

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ZmWUS (see US20110167516, published July 7, 2011) were mixed with a particle delivery matrix comprising commercially available gold particles (0.6pm, Bio-Rad) and a water soluble cationic lipid TranslT-2020 (Mirus, USA). The particle delivery matrix comprising the guide RNA/Cas endonuclease ribonucleotide-protein complexes were delivered into maize embryo cells using particle mediated delivery (see Particle-mediated delivery described in Example 8) with some modifications. Specifically, after gold particles were pelleted in a microfuge at 10,000 rpm for 1 min and supernatant was removed, the particles were resuspended in 105 pi of sterile water instead of 100% ethanol. Then, 10 pi was spotted onto the center of each io macrocarrier and allowed to dry before bombardment.

Wild type maize plants were used as a source of immature embryos for codelivery of Cas9 and gRNA in the form of a guide RNA/Cas endonuclease ribonucleotide-protein complexes (RGEN) along with selectable and visible marker (UBI:MoPAT-DsRED) and developmental genes (UBI:ZmODP2 and IN2:ZmWUS).

To measure mutation frequencies at the LIGCas-3, MS26Cas-2, MS45Cas-2 and ALSCas-4 endogenous target sites, embryos were harvested two days postbombardment and analyzed by amplicon deep sequencing. Untreated embryos and embryos bombarded with the Cas9 protein only served as negative controls while embryos bombarded with DNA vectors expressing Cas9 and gRNA were used as positive controls. Similar frequencies were detected for Cas9-gRNA components delivered as DNA vectors and as guide RNA/Cas endonuclease ribonucleotideprotein complexes (Table 11).

Table 11. Percentage of mutant reads at LIG, MS26, MS45, and ALS target sites produced by direct delivery of RGEN complexes into maize embryo cells.

Target Sites	Molecules delivered	Total Number of Reads	Number of Mutant Reads	Percentage of Mutant Reads
LIGCas-3 (Chr. 2)	Untreated embryos (control 1)	915,198	38	0.004%
Cas9 protein only (control 2)	408,348	17	0.004%
Cas9-Lig-CR3 RGEN	439,827	2,510	0.57%

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	Cas9 (DNA)+Lig-CR3 (DNA)	369,443	2,058	0.56%
MS26Cas-2 (Chr. 1)	Untreated embryos (control 1)	245,476	8	0.003%
Cas9 protein only (control 2)	429,388	20	0.004%
Cas9-MS26-CR2 RGEN	252,519	533	0.21%
Cas9 (DNA)+MS26-CR2 (DNA)	186,857	812	0.43%
MS45Cas-2 (Chr. 9)	Untreated embryos (control 1)	255,877	12	0.005%
Cas9 protein only (control 2)	487,876	12	0.002%
Cas9-MS45-CR2 RGEN	241,287	2,075	0.86%
Cas9 (DNA)+MS45-CR2 (DNA)	304,622	1591	0.52%
ALS2Cas-4 (Chr. 5)	Cas9 protein only (control 2)	807,014	125	0.02%
Cas9-ALS-CR4 RGEN	791,084	3,613	0.45%
Cas9 (DNA)+ALS-CR4 (DNA)	833,130	4251	0.51%

To measure the mutation frequency at the plant level, 60 embryos cobombarded with Cas9-MS45-gRNA complex, ZmODP2, ZmWUS and MOPATDSRED were placed on media containing bialaphos as selective agent. Multiple plants were regenerated from each of the 36 herbicide-resistant callus sectors and screened for mutations. Out of the 36 events, 17 (47%) contained mutant alleles (10 single and 7 biallelic) while 19 (53%) revealed only wild type MS45 alleles. Among plants with mutations, the number of sequencing reads for each allele was similar indicating plants were not chimeric.

io To demonstrate that direct RGEN delivery is also sufficient to generate specific edits in endogenous genes in plants, the maize ALS2 gene was targeted (ALS2-specific ALSCas-4 target site) as described in Example 2. A 127 nt singlestranded DNA Oligo2, (SEQ ID NO: 45) as a repair template was co-delivered with Cas9/ALS-CR4 RGEN complex in a similar manner as described above. Embryos were harvested two days post-bombardment and analyzed by amplicon deep sequencing (Table 12).

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Table 12. Percentage of mutant reads and reads with edits at ALS target site produced by delivery of RGEN complex and donor DNA template into maize embryo cells.

Target Site	Molecules delivered	Total Number of Reads	Number of Mutant Reads	% of Mutant Reads	Number of Reads with Edits	% of Reads with Edits
ALS2	Cas9 protein only	807,014	105	0.01%	-	-
Cas9-ALSCR4 RGEN + ss Oligo2	791,084	3,613	0.45%	209	0.02%

In addition, in two independent experiments, 40 to 50 bombarded embryos were transferred to plates containing 100 ppm of chlorsulfuron as direct selection for an edited ALS2 gene. Six weeks later, two callus sectors (one from each experiment) that continued growing on media with chlorsulfuron were analyzed by sequencing. In both events, one ALS2 allele was specifically edited while the io second allele remained wild type. Plants regenerated from these callus sectors contained edited ALS2 alleles and were resistant to chlorsulfuron when sprayed with the herbicide.

These data demonstrate that direct delivery of Cas9 and gRNA, in the form of a guide RNA/Cas endonuclease ribonucleotide-protein complex (with or without a polynucleotide modification template DNA) into maize immature embryo cells, is a viable alternative to DNA delivery (such as recombinant DNA, plasmid DNA) for targeted mutagenesis and gene editing in plants.

EXAMPLE 11

Direct Delivery of Cas9 in the form on mRNA and gRNA into Embryo Cells

Generates Mutations in Maize

This example demonstrates that direct delivery of Cas9, in the form of mRNA molecules and gRNA in the form of in vitro transcribed or chemically synthesized RNA molecules, into maize immature embryo cells generates mutations at the corresponding targeted sites.

In our previous experiment (Svitashev etal., Plant Physiology, 2015, Vol.

169, pp. 931-945), co-delivery of gRNA in the form of in vitro synthesized RNA

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PCT/US2016/057279 molecules with the Cas9 expressing DNA vector yielded approximately 100-fold lower mutation frequency then in experiments where both Cas9 and gRNA were delivered as DNA vectors. One possible explanation for this difference may be that the requirement for coincident function of Cas9 and gRNA was not met when gRNA was delivered as RNA and Cas9 was delivered as a DNA vector. To overcome this problem, Cas9 can be delivered as mRNA molecules that will shorten the time from the moment of delivery to functional Cas9 protein expression. Commercially available Cas9 mRNA (TriLink Biotechnologies) was used in the experiment.

To test this idea, maize embryo cells were co-bombarded with Cas9 mRNA (200ng), gRNA in the form of in vitro synthesized RNA molecules (100ng), DNA vectors containing Ubiquitin regulated MoPAT-DsRED fusion (25ng) and developmental genes, Ubiquitin promoter regulated ODP2 and IN2 promoter regulated WUS (12ng each) per shot. Commercially available Cas9 mRNA (TriLink Biotechnologies) and RNA molecules, in vitro synthesized as described above, were used in the experiment. Analysis for mutation frequency was performed by amplicon deep sequencing on embryos collected 2 days post-transformation (Table 13).

Table 13. Percentage of mutant reads at MS45 target site produced by transient delivery of Cas9 as mRNA and gRNA as RNA molecules into maize embryo cells.

Target Sites	Molecules delivered	Total Number of Reads	Number of Mutant Reads	Percentage of Mutant Reads
MS45	Cas9 mRNA only	1,097,279	799	0.07%
Cas9 mRNA+Ms45 CR2 gRNA	1,260,332	2,304	0.18%
Cas9 (DNA)+Ms45 CR2 (DNA)	1,106,125	3,490	0.31%

These data demonstrate that delivery of both Cas9 and gRNA, in the form of RNA molecules, improves frequencies of targeted mutations and, along with Cas9gRNA delivery as the RGEN complex, is a viable alternative to DNA delivery for targeted mutagenesis and gene editing in plants.

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EXAMPLE 12

Direct Delivery of Cas9 and gRNA as a guide RNA/Cas endonuclease Ribonucleotide-protein Complex (RGEN) into Embryo Cells without the Use of a

Selectable Marker Generates Mutations in Maize

This example demonstrates that delivery of Cas9 in the form of protein and gRNA in the form of in vitro transcribed or chemically synthesized RNA molecules, into maize immature embryo cells without co-delivery of selectable marker gene(s) is sufficient to regenerate plants with mutations at the corresponding targeted sites with practical frequencies.

io The necessity of selectable markers to provide a growth advantage to transformed or modified cells has been the long standing paradigm in plant transformation and genome modification protocols. Therefore, in all mutation, gene editing and gene integration experiments, selectable markers are used to select for genome edited events. Taking into consideration the unexpected high activity (mutation frequency) of RGEN complexes in the experiments described in Example 10, a completely DNA-free (vector free) genome editing without a selectable marker was attempted. Maize embryo cells were bombarded with guide RNA/Cas endonuclease ribonucleotide-protein (RGEN) complexes targeting three different genes: ligulelessl (LIG), MS26 and MS45. Cas9 endonuclease and MS45-gRNA on

DNA vectors were delivered in parallel experiments which served as controls. Plants were regenerated and analyzed by sequencing for targeted mutations. In all experiments, mutant plants were recovered at surprisingly high frequencies ranging from 2.4 to 9.7% (Table 14).

Table 14. Mutation frequencies at LIG, MS26 and MS45 target sites upon delivery of

Cas9 and gRNAs in the form of DNA vectors and direct delivery of RGEN complexes into maize immature embryo cells. Analysis was performed on TO plants regenerated without selection (without use of selectable markers).

Target site	Cas9 and gRNA delivery method	Plants analyzed	Plants with mutated alleles	SEQ ID NO:
LIGCas-3	RGEN	756	73 (9.7%)	12
MS26Cas-2	RGEN	756	18 (2.4%)	14

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MS45 Cas-2	RGEN	1,880	70 (3.7%)	8
MS45 Cas-2	Vector DNA	940	38 (4.0%)	8

Further, regenerated TO plants were crossed with wild type Hi-Il plants and the progeny plants were used for segregation analysis. Sexual transmission of mutated ms45 alleles at the expected Mendelian segregation (1:1) was demonstrated in all progeny plants analyzed.

To assess the potential of RGEN delivery to reduce off-target cleavage in maize, the mutation frequency at the MS45 off-site was evaluated using DNA vectors and RGEN delivery. Searches for a site with close homology to the on-target site were performed by aligning the protospacer region of the MS45Cas-2 target site io (the region of the target site that base pairs with the guide RNA spacer) with the maize B73 reference genome (B73 RefGen_v3, Maize Genetics and Genomics Database) using Bowtie sequence aligner (Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10:R25, 2009) permitting up to two mismatches with the on-target sequence. Potential off-target sites were then examined for the presence of a NGG protospacer adjacent motif (PAM) sequence immediately 3’ of the identified protospacer off-target. Only a single off-target site (5’CGCCGAGGGCGACTACCGGC-3’, SEQ ID NO:81) was identified using these search criteria. It contained a 2 bp mismatch with the MS45 protospacer target and an AGG PAM (Table 15). To confirm the site was cleaved in vivo, it was analyzed by deep sequencing for the presence of mutations in maize embryos transformed with DNA vectors expressing Cas9 and MS45-CR2 gRNA. As shown in Table 15, mutational activity of the off-target site was at a frequency of 2% compared to a 4% frequency observed for the on-target site. As shown in Table 15, RGEN off-target activity was greatly reduced relative to Cas9 and gRNA delivery on DNA vectors (from 2% to 0%).

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Table 15. Mutation frequencies at the MS45 off-target site upon delivery of Cas9 and gRNAs in the form of DNA vectors and RGEN complexes into maize immature embryo cells. Analysis performed on TO plants regenerated without selection.

Target Site	Cas9 and gRNA delivery method	Target site sequence with PAM*	Plants Analyzed	Plants with Mutations (number)	Plants with Mutation s (%)	SEQ ID NO:
MS45Cas- 2		GGCCGAGGTCGACTAC CGGCCGG	904	38	4%	14
MS45 offsite	DNA	CGCCGAGGGCGACTAC CGGCAGG	940	19	2.0%	82
MS45 offsite	RGEN	CGCCGAGGGCGACTAC CGGCAGG	1,880	0	0.0%	82

*PAM - protospacer adjacent motif is a 3 nt sequence immediately 3’ of the target site.

Two nucleotides different in the MS45 off-target site in comparison to the intended site are shown in bold and underlined.

This example demonstrates that generation of plants with targeted mutations io using RNA-guided endonucleases, does not require co-delivery of selectable or screenable marker genes, thus increasing specificity and exactness of the introduced modifications. Regenerated plants contained only targeted mutations or targeted gene edits (if a repair template was included to modify DNA sequence) without random integration of DNA vectors. This method of delivery provides a completely DNA-free approach to gene mutagenesis in plant cells of major crop species including but not limited to maize, soybean, wheat, rice, millet, sorghum and canola.

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THAT WHICH

Claims (7)

1. IS CLAIMED:

   1. A method for restoring function to a non-functional gene product in the genome of a cell, the method comprising introducing a guide RNA/Cas endonuclease complex into a cell comprising a disrupted gene in its genome, wherein said

   5 complex creates a double strand break, wherein said disrupted gene does not encode a functional gene product, wherein said disrupted gene is restored without the use of a polynucleotide modification template to a non-disrupted gene capable of encoding said functional gene product.

   io 2. The method of claim 1, wherein said disrupted gene comprises a base pair deletion of the 4^th nucleotide upstream (5’) of a PAM sequence when compared to its corresponding non-disrupted gene, wherein said base pair deletion creates an amino acid frameshift in the gene product of the disrupted gene thereby rendering the gene product of the disrupted gene non-functional.

   3. The method of claim 2, wherein the base pair deletion is the first, second, or third nucleotide of a codon sequence.

   4. The method of claim 1, wherein the restoration is accomplished by Non20 Homologous-End -Joining (NHEJ) resulting in the insertion of a single base into the double strand break.

   5. A method for modifying a nucleotide sequence in the genome of a cell, the method comprising:

   25 introducing into at least one cell comprising a target site and a disrupted selectable marker gene, a first guide RNA, a Cas endonuclease, and at least a second guide RNA, wherein said first guide RNA and Cas endonuclease can form a first complex capable of introducing a double strand break in said disrupted selectable marker gene, wherein said disrupted selectable marker gene is restored

   30 without the use of a polynucleotide modification template to a non-disrupted selectable marker gene capable of encoding a functional selectable marker protein, wherein said second guide RNA and Cas endonuclease can form a second complex

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   PCT/US2016/057279 that is capable of recognizing, binding to, and nicking or cleaving said target site located in said nucleotide sequence; and, selecting a cell having a modification in said nucleotide sequence, wherein the selection is provided by said functional selectable marker protein.

   6. The method of claim 5, wherein the introducing and selection step does not comprise the introduction of a selectable marker gene.

   7. The method of claim 5, wherein the modification is selected from the group io consisting of an insertion of at least one nucleotide, a deletion of at least one nucleotide, and a substitution of at least one nucleotide in said target site.

   8. The method of claim 5, further comprising introducing a polynucleotide modification template into said cell, wherein said polynucleotide modification template

   15 comprises at least one nucleotide modification of said nucleotide sequence.

   9. The method of claim 8, wherein the at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii)

   20 an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii).

   10. The method of claim 5, further comprising introducing a donor DNA into said cell, wherein said donor DNA comprises at least one polynucleotide of interest to be inserted into said target site.

   11. The method of claim 5, wherein the cell is selected from the group consisting of a human, non-human, animal, archaea, bacterial, fungal, insect, yeast, nonconventional yeast, and plant cell.

   30 12. The method of claim 11, wherein the plant cell is selected from the group consisting of a monocot and dicot cell.

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   13. The method of claim 11, wherein the plant cell is selected from the group consisting of a maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco, Arabidopsis, and safflower cell.

   14. The method of claim 11, further comprising producing a plant or progeny plant from said plant cell.

   15. A plant or progeny plant produced by the method of claim 14, wherein said io plant or progeny plant is void of any one guide RNA and Cas endonuclease.

   16. A method for editing a nucleotide sequence in the genome of a cell without the use of a polynucleotide modification template, the method comprising:

   a) introducing into at least one cell at least one guide RNA and at least one Cas

   15 endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence;

   b) selecting a cell from (a) comprising at least one single nucleotide deletion in said nucleotide sequence, wherein said nucleotide deletion is located at a

   20 position to be edited; and,

   c) introducing into a cell of (b) at least one guide RNA and at least one Cas endonuclease, wherein said guide RNA and Cas endonuclease can form a complex capable of introducing a double strand break in said nucleotide sequence and insert a single nucleotide at the same position of the nucleotide

   25 deletion of (b) without the use of a polynucleotide modification template.

   17. The method of claim 1, wherein the guide RNA and Cas endonuclease protein forming the guide RNA /Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

   18. The method of claim 1, wherein the guide RNA /Cas endonuclease complex is introduced into the cell as a ribonucleotide-protein complex.

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   19. The method of claim 1, wherein components of the guide RNA/Cas endonuclease complex are introduced as mRNA encoding the Cas endonuclease protein and as RNA comprising the guide RNA.

   20. A method of delivering a guide RNA /Cas endonuclease complex into a cell, the method comprising combining at least one guide RNA molecule and at least one Cas endonuclease protein to form a ribonucleotide-protein and combining said io ribonucleotide-protein with a particle delivery matrix to allow for said ribonucleotideprotein and matrix to bind and form a ribonucleotide-protein-matrix complex; and, introducing said ribonucleotide-protein-matrix complex into said cell.

   21. The method of claim 20, further comprising introducing a polynucleotide

   15 template, wherein said polynucleotide modification template comprises at least one nucleotide modification of a nucleotide sequence in the genome of said cell, wherein said at least one nucleotide modification of said polynucleotide modification template is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one

   20 nucleotide, and (iv) any combination of (i) - (iii).

   22. The method of claim 20, further comprising introducing a donor DNA, wherein said donor DNA comprises at least one polynucleotide of interest.

   25 23. The method of claim 20, wherein the particle delivery matrix comprises a microparticle combined with a cationic lipid.

   24. The method of claim 1, wherein said Cas endonuclease is selected from the group consisting of a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein,

   30 a C2c3 protein, Cas3, Cas3-H, Cas 5, Cas7, Cas8, Casio, or complexes of these.

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   20161011_BB2533PCT_SeqLst.txt SEQUENCE LISTING <110> Pioneer Hi-Bred International Inc.

   Cigan, Andrew Mark Svitashev, Sergei <120> RESTORING FUNCTION TO A NON-FUNCTIONAL GENE PRODUCT VIA GUIDED CAS SYSTEMS AND METHODS OF USE <130> BB2533 PCT <150> 62/243719 <151> 2015-10-20 <150> 62/309033 <151> 2016-03-16 <150> 62/359254 <151> 2016-07-07 <160> 82 <170> PatentIn version 3.5 <210> 1 <211> 4107 <212> DNA <213> Streptococcus pyogenes M1 GAS (SF370) <400> 1 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280

   Page 1

   20161011_BB2533PCT_SeqLst.txt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg tgactga <210> 2 <211> 189 <212> DNA <213> Solanum tuberosum <400> 2 gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaacatggt gatgtgcag <210> 3 <211> 9 <212> PRT <213> Simian virus 40 <400> 3

   Met Ala Pro Lys Lys Lys Arg Lys Val

   1 5 <210> 4 <211> 18 <212> PRT <213> Agrobacterium tumefaciens <400> 4

   Lys Arg Pro Arg Asp Arg His Asp Gly Glu Leu Gly Gly Arg Lys Arg 1 5 10 15

   Ala Arg

   2340

   2400

   2460

   2520

   2580

   2640

   2700

   2760

   2820

   2880

   2940

   3000

   3060

   3120

   3180

   3240

   3300

   3360

   3420

   3480

   3540

   3600

   3660

   3720

   3780

   3840

   3900

   3960

   4020

   4080

   4107

   120

   180

   189 <210> 5 <211> 6717 <212> DNA <213> Artificial Sequence

   Page 2

   20161011_BB2533PCT_SeqLst.txt <220>

   <223> Maize optimized Cas9 expression cassette <400> 5 gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta 60 taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt 120 atacatatat ttaaacttta ctctacgaat aatataatct atagtactac aataatatca 180 gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt 240 ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg 300 caaatagctt cacctatata atacttcatc cattttatta gtacatccat ttagggttta 360 gggttaatgg tttttataga ctaatttttt tagtacatct attttattct attttagcct 420 ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa 480 tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta 540 aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt 600 ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca 660 cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg 720 ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag 780 gcggcctcct cctcctctca cggcaccggc agctacgggg gattcctttc ccaccgctcc 840 ttcgctttcc cttcctcgcc cgccgtaata aatagacacc ccctccacac cctctttccc 900 caacctcgtg ttgttcggag cgcacacaca cacaaccaga tctcccccaa atccacccgt 960 cggcacctcc gcttcaaggt acgccgctcg tcctcccccc cccccctctc taccttctct 1020 agatcggcgt tccggtccat gcatggttag ggcccggtag ttctacttct gttcatgttt 1080 gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg gatgcgacct 1140 gtacgtcaga cacgttctga ttgctaactt gccagtgttt ctctttgggg aatcctggga 1200 tggctctagc cgttccgcag acgggatcga tttcatgatt ttttttgttt cgttgcatag 1260 ggtttggttt gcccttttcc tttatttcaa tatatgccgt gcacttgttt gtcgggtcat 1320 cttttcatgc ttttttttgt cttggttgtg atgatgtggt ctggttgggc ggtcgttcta 1380 gatcggagta gaattctgtt tcaaactacc tggtggattt attaattttg gatctgtatg 1440 tgtgtgccat acatattcat agttacgaat tgaagatgat ggatggaaat atcgatctag 1500 gataggtata catgttgatg cgggttttac tgatgcatat acagagatgc tttttgttcg 1560 cttggttgtg atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga 1620 atactgtttc aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac 1680 atcttcatag ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt 1740 tgatgtgggt tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc 1800 taaccttgag tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg 1860 atatacttgg atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca 1920 tacgctattt atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt 1980 tacttctgca ggtcgactct agaggatcca tggcaccgaa gaagaagcgc aaggtgatgg 2040 acaagaagta cagcatcggc ctcgacatcg gcaccaactc ggtgggctgg gccgtcatca 2100 cggacgaata taaggtcccg tcgaagaagt tcaaggtcct cggcaataca gaccgccaca 2160 gcatcaagaa aaacttgatc ggcgccctcc tgttcgatag cggcgagacc gcggaggcga 2220 ccaggctcaa gaggaccgcc aggagacggt acactaggcg caagaacagg atctgctacc 2280 tgcaggagat cttcagcaac gagatggcga aggtggacga ctccttcttc caccgcctgg 2340 aggaatcatt cctggtggag gaggacaaga agcatgagcg gcacccaatc ttcggcaaca 2400 tcgtcgacga ggtaagtttc tgcttctacc tttgatatat atataataat tatcattaat 2460 tagtagtaat ataatatttc aaatattttt ttcaaaataa aagaatgtag tatatagcaa 2520 ttgcttttct gtagtttata agtgtgtata ttttaattta taacttttct aatatatgac 2580 caaaacatgg tgatgtgcag gtggcctacc acgagaagta cccgacaatc taccacctcc 2640 ggaagaaact ggtggacagc acagacaagg cggacctccg gctcatctac cttgccctcg 2700 cgcatatgat caagttccgc ggccacttcc tcatcgaggg cgacctgaac ccggacaact 2760 ccgacgtgga caagctgttc atccagctcg tgcagacgta caatcaactg ttcgaggaga 2820 accccataaa cgctagcggc gtggacgcca aggccatcct ctcggccagg ctctcgaaat 2880 caagaaggct ggagaacctt atcgcgcagt tgccaggcga aaagaagaac ggcctcttcg 2940 gcaaccttat tgcgctcagc ctcggcctga cgccgaactt caaatcaaac ttcgacctcg 3000 cggaggacgc caagctccag ctctcaaagg acacctacga cgacgacctc gacaacctcc 3060 tggcccagat aggagaccag tacgcggacc tcttcctcgc cgccaagaac ctctccgacg 3120 ctatcctgct cagcgacatc cttcgggtca acaccgaaat taccaaggca ccgctgtccg 3180 ccagcatgat taaacgctac gacgagcacc atcaggacct cacgctgctc aaggcactcg 3240 tccgccagca gctccccgag aagtacaagg agatcttctt cgaccaatca aaaaacggct 3300 acgcgggata tatcgacggc ggtgccagcc aggaagagtt ctacaagttc atcaaaccaa 3360 tcctggagaa gatggacggc accgaggagt tgctggtcaa gctcaacagg gaggacctcc 3420 tcaggaagca gaggaccttc gacaacggct ccatcccgca tcagatccac ctgggcgaac 3480 tgcatgccat cctgcggcgc caggaggact tctacccgtt cctgaaggat aaccgggaga 3540 agatcgagaa gatcttgacg ttccgcatcc catactacgt gggcccgctg gctcgcggca 3600 actcccggtt cgcctggatg acccggaagt cggaggagac catcacaccc tggaactttg 3660 aggaggtggt cgataagggc gctagcgctc agagcttcat cgagcgcatg accaacttcg 3720 ataaaaacct gcccaatgaa aaagtcctcc ccaagcactc gctgctctac gagtacttca 3780 ccgtgtacaa cgagctcacc aaggtcaaat acgtcaccga gggcatgcgg aagccggcgt 3840

   Page 3

   20161011_BB2533PCT_SeqLst.txt tcctgagcgg cgagcagaag aaggcgatag tggacctcct cttcaagacc aacaggaagg 3900 tgaccgtgaa gcaattaaaa gaggactact tcaagaaaat agagtgcttc gactccgtgg 3960 agatctcggg cgtggaggat cggttcaacg cctcactcgg cacgtatcac gacctcctca 4020 agatcattaa agacaaggac ttcctcgaca acgaggagaa cgaggacatc ctcgaggaca 4080 tcgtcctcac cctgaccctg ttcgaggacc gcgaaatgat cgaggagagg ctgaagacct 4140 acgcgcacct gttcgacgac aaggtcatga aacagctcaa gaggcgccgc tacactggtt 4200 ggggaaggct gtcccgcaag ctcattaatg gcatcaggga caagcagagc ggcaagacca 4260 tcctggactt cctcaagtcc gacgggttcg ccaaccgcaa cttcatgcag ctcattcacg 4320 acgactcgct cacgttcaag gaagacatcc agaaggcaca ggtgagcggg cagggtgact 4380 ccctccacga acacatcgcc aacctggccg gctcgccggc cattaaaaag ggcatcctgc 4440 agacggtcaa ggtcgtcgac gagctcgtga aggtgatggg ccggcacaag cccgaaaata 4500 tcgtcataga gatggccagg gagaaccaga ccacccaaaa agggcagaag aactcgcgcg 4560 agcggatgaa acggatcgag gagggcatta aagagctcgg gtcccagatc ctgaaggagc 4620 accccgtgga aaatacccag ctccagaatg aaaagctcta cctctactac ctgcagaacg 4680 gccgcgacat gtacgtggac caggagctgg acattaatcg gctatcggac tacgacgtcg 4740 accacatcgt gccgcagtcg ttcctcaagg acgatagcat cgacaacaag gtgctcaccc 4800 ggtcggataa aaatcggggc aagagcgaca acgtgcccag cgaggaggtc gtgaagaaga 4860 tgaaaaacta ctggcgccag ctcctcaacg cgaaactgat cacccagcgc aagttcgaca 4920 acctgacgaa ggcggaacgc ggtggcttga gcgaactcga taaggcgggc ttcataaaaa 4980 ggcagctggt cgagacgcgc cagatcacga agcatgtcgc ccagatcctg gacagccgca 5040 tgaatactaa gtacgatgaa aacgacaagc tgatccggga ggtgaaggtg atcacgctga 5100 agtccaagct cgtgtcggac ttccgcaagg acttccagtt ctacaaggtc cgcgagatca 5160 acaactacca ccacgcccac gacgcctacc tgaatgcggt ggtcgggacc gccctgatca 5220 agaagtaccc gaagctggag tcggagttcg tgtacggcga ctacaaggtc tacgacgtgc 5280 gcaaaatgat cgccaagtcc gagcaggaga tcggcaaggc cacggcaaaa tacttcttct 5340 actcgaacat catgaacttc ttcaagaccg agatcaccct cgcgaacggc gagatccgca 5400 agcgcccgct catcgaaacc aacggcgaga cgggcgagat cgtctgggat aagggccggg 5460 atttcgcgac ggtccgcaag gtgctctcca tgccgcaagt caatatcgtg aaaaagacgg 5520 aggtccagac gggcgggttc agcaaggagt ccatcctccc gaagcgcaac tccgacaagc 5580 tcatcgcgag gaagaaggat tgggacccga aaaaatatgg cggcttcgac agcccgaccg 5640 tcgcatacag cgtcctcgtc gtggcgaagg tggagaaggg caagtcaaag aagctcaagt 5700 ccgtgaagga gctgctcggg atcacgatta tggagcggtc ctccttcgag aagaacccga 5760 tcgacttcct agaggccaag ggatataagg aggtcaagaa ggacctgatt attaaactgc 5820 cgaagtactc gctcttcgag ctggaaaacg gccgcaagag gatgctcgcc tccgcaggcg 5880 agttgcagaa gggcaacgag ctcgccctcc cgagcaaata cgtcaatttc ctgtacctcg 5940 ctagccacta tgaaaagctc aagggcagcc cggaggacaa cgagcagaag cagctcttcg 6000 tggagcagca caagcattac ctggacgaga tcatcgagca gatcagcgag ttctcgaagc 6060 gggtgatcct cgccgacgcg aacctggaca aggtgctgtc ggcatataac aagcaccgcg 6120 acaaaccaat acgcgagcag gccgaaaata tcatccacct cttcaccctc accaacctcg 6180 gcgctccggc agccttcaag tacttcgaca ccacgattga ccggaagcgg tacacgagca 6240 cgaaggaggt gctcgatgcg acgctgatcc accagagcat cacagggctc tatgaaacac 6300 gcatcgacct gagccagctg ggcggagaca agagaccacg ggaccgccac gatggcgagc 6360 tgggaggccg caagcgggca aggtaggtac cgttaaccta gacttgtcca tcttctggat 6420 tggccaactt aattaatgta tgaaataaaa ggatgcacac atagtgacat gctaatcact 6480 ataatgtggg catcaaagtt gtgtgttatg tgtaattact agttatctga ataaaagaga 6540 aagagatcat ccatatttct tatcctaaat gaatgtcacg tgtctttata attctttgat 6600 gaaccagatg catttcatta accaaatcca tatacatata aatattaatc atatataatt 6660 aatatcaatt gggttagcaa aacaaatcta gtctaggtgt gttttgcgaa tgcggcc 6717 <210> 6 <211> 4437 <212> DNA <213> Artificial sequence <220>

   <223> Lig-CR3 guide RNA expression vector <400> 6 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 60 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 120 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 180 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 240 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 300 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 360 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 420 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca 480 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 540 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 600

   Page 4

   20161011_BB2533PCT_SeqLst.txt aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 660 ccctcgaggt cgacggtatc gataagcttt gagagtacaa tgatgaacct agattaatca 720 atgccaaagt ctgaaaaatg caccctcagt ctatgatcca gaaaatcaag attgcttgag 780 gccctgttcg gttgttccgg attagagccc cggattaatt cctagccgga ttacttctct 840 aatttatata gattttgatg agctggaatg aatcctggct tattccggta caaccgaaca 900 ggccctgaag gataccagta atcgctgagc taaattggca tgctgtcaga gtgtcagtat 960 tgcagcaagg tagtgagata accggcatca tggtgccagt ttgatggcac cattagggtt 1020 agagatggtg gccatgggcg catgtcctgg ccaactttgt atgatatatg gcagggtgaa 1080 taggaaagta aaattgtatt gtaaaaaggg atttcttctg tttgttagcg catgtacaag 1140 gaatgcaagt tttgagcgag ggggcatcaa agatctggct gtgtttccag ctgtttttgt 1200 tagccccatc gaatccttga cataatgatc ccgcttaaat aagcaacctc gcttgtatag 1260 ttccttgtgc tctaacacac gatgatgata agtcgtaaaa tagtggtgtc caaagaattt 1320 ccaggcccag ttgtaaaagc taaaatgcta ttcgaatttc tactagcagt aagtcgtgtt 1380 tagaaattat ttttttatat accttttttc cttctatgta cagtaggaca cagtgtcagc 1440 gccgcgttga cggagaatat ttgcaaaaaa gtaaaagaga aagtcatagc ggcgtatgtg 1500 ccaaaaactt cgtcacagag agggccataa gaaacatggc ccacggccca atacgaagca 1560 ccgcgacgaa gcccaaacag cagtccgtag gtggagcaaa gcgctgggta atacgcaaac 1620 gttttgtccc accttgacta atcacaagag tggagcgtac cttataaacc gagccgcaag 1680 caccgaattg cgtacgcgta cgtgtggttt tagagctaga aatagcaagt taaaataagg 1740 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt tgcggccgcg 1800 aattcctgca gggccctctt gtcggaccag ttgcccacca cgttggtgag ctcggtgagg 1860 cccttcattg agaggaagga ggtcatgagg tgcctaccga tgtgggactt ggggccgttc 1920 ttgatggcga agatggagta gggggcgttc ttcttgaggg ccttgttgta ggacctcacg 1980 aggttgtcct tgaggagctg gtactcctgc ttgttggagg aggagttgcc ggtcctgttc 2040 accctcttga gcacgggctc tgagttcctg aggaactcgt cgaggtacac gagggggtcg 2100 atcctgccgc gagcggagaa gaagtagatg tgcctggaca cggaggtctt ggtctcggtc 2160 acgaggcact ggatgatcac gccgaggtac ttgttctgac tagttctaga gcggccgcca 2220 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa 2280 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 2340 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 2400 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 2460 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 2520 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 2580 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2640 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2700 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2760 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2820 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2880 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2940 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3000 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3060 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3120 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3180 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3240 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3300 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3360 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3420 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3480 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3540 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3600 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3660 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3720 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 3780 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3840 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3900 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3960 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4020 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 4080 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4140 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4200 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4260 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4320 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4380 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacct 4437 <210> 7 <211> 27 <212> DNA

   Page 5

   20161011_BB2533PCT_SeqLst.txt <213> Zea mays

   <400> 7 gtactccatc cgccccatcg agtaggg <210> 8 27 <211> <212> <213> 24 DNA Zea mays <400> 8 gcacgtacgt caccatcccg ccgg 24 <210> 9 <211> 24 <212> DNA <213> Zea mays <400> 9 gacgtacgtg ccctactcga tggg 24 <210> 10 <211> 24 <212> DNA <213> Zea mays <400> 10 gtaccgtacg tgccccggcg gagg 24 <210> 11 <211> 24 <212> DNA <213> Zea mays <400> 11 ggaattgtac cgtacgtgcc ccgg 24 <210> 12 <211> 20 <212> DNA <213> Zea mays <400> 12 gcgtacgcgt acgtgtgagg 20 <210> 13 <211> 22 <212> DNA <213> Zea mays <400> 13 gctggccgag gtcgactacc gg 22 <210> 14 <211> 23 <212> DNA <213> Zea mays <400> 14 ggccgaggtc gactaccggc cgg 23 <210> 15 <211> 23 <212> DNA <213> Zea mays <400> 15 ggcgcgagct cgtgcttcac cgg 23

   Page 6

   20161011_BB2533PCT_SeqLst.txt <210> 16 <211> 21 <212> DNA <213> Zea mays <400> 16 ggtgccaatc atgcgtcgcg g 21 <210> 17 <211> 20 <212> DNA <213> Zea mays <400> 17 ggtcgccatc acgggacagg 20 <210> 18 <211> 24 <212> DNA <213> Zea mays <400> 18 gtcgcggcac ctgtcccgtg atgg 24 <210> 19 <211> 56 <212> DNA <213> Artificial Sequence <220>

   <223> MS26Cas-1 forward primer <400> 19 ctacactctt tccctacacg acgctcttcc gatctaggac cggaagctcg ccgcgt 56 <210> 20 <211> 54 <212> DNA <213> Artificial Sequence <220>

   <223> MS26Cas-1 and MS26Cas-3 reverse primer

   <400> caagca 20 54 gaag acggcatacg agctcttccg atcttcctgg aggacgacgt gctg <210> 21 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> MS26Cas-2 forward primer <400> 21 ctacactctt tccctacacg acgctcttcc gatctaaggt cctggaggac gacgtgctg 59 <210> 22 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> MS26Cas-2 reverse primer <400> 22 caagcagaag acggcatacg agctcttccg atctccggaa gctcgccgcg t 51 Page 7

   20161011_BB2533PCT_SeqLst.txt <210> 23 <211> 56 <212> DNA <213> Artificial Sequence <220>

   <223> MS26Cas-3 forward primer <400> 23

   ctacactctt tccctacacg acgctcttcc gatcttcctc cggaagctcg ccgcgt 56 <210> 24 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> LIGCas-1 forward primer <400> 24 ctacactctt tccctacacg acgctcttcc gatctaggac ctg <210> 25 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> LIGCas-1 and LIGCas-2 reverse primer <400> 25 tgtaacgatt tacgcacctg 60 63 caagcagaag acggcatacg agctcttccg atctgcaaat <210> 26 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> LIGCas-2 forward primer <400> 26 gagtagcagc gcacgtat 58 ctacactctt tccctacacg acgctcttcc gatcttcctc ctg <210> 27 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> LIGCas-3 forward primer <400> 27 tgtaacgatt tacgcacctg 60 63 ctacactctt tccctacacg acgctcttcc gatctaaggc <210> 28 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> LIGCas-3 reverse primer <400> 28 gcaaatgagt agcagcgcac 60 caagcagaag acggcatacg agctcttccg atctcacctg Page 8 ctgggaattg taccgta 57

   20161011_BB2533PCT_SeqLst.txt <210> 29 <211> 58 <212> DNA <213> Artificial Sequence <220>

   <223> MS45Cas-1 forward primer <400> 29 ctacactctt tccctacacg acgctcttcc gatctaggag gacccgttcg gcctcagt 58 <210> 30 <211> 54 <212> DNA <213> Artificial Sequence <220>

   <223> S45Cas-1, MS45Cas-2 and MS45Cas-3 reverse primer <400> 30

   caagcagaag acggcatacg agctcttccg atctgccggc tggcattgtc tctg 54 <210> 31 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> MS45Cas-2 forward primer <400> 31 ctacactctt tccctacacg acgctcttcc gatcttcctg gacccgttcg gcctcagt 58 <210> 32 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> MS45Cas-3 forward primer <400> 32 ctacactctt tccctacacg acgctcttcc gatctgaagg gacccgttcg gcctcagt 58 <210> 33 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> ALSCas-1 forward primer <400> 33 ctacactctt tccctacacg acgctcttcc gatctaaggc gacgatgggc gtctcctg 58 <210> 34 <211> 53 <212> DNA <213> Artificial Sequence

   <220>

   <223> ALSCas-1, ALSCas-2 and ALSCas-3 reverse primer <400> 34 caagcagaag acggcatacg agctcttccg atctgcgtct gcatcgccac ctc 53 <210> 35

   Page 9

   20161011_BB2533PCT_SeqLst.txt <211> 58 <212> DNA <213> Artificial Sequence <220>

   <223> ALSCas-2 forward primer <400> 35 ctacactctt tccctacacg acgctcttcc gatctttccc gacgatgggc gtctcctg 58 <210> 36 <211> 58 <212> DNA <213> Artificial Sequence <220>

   <223> ALSCas-3 forward primer <400> 36 ctacactctt tccctacacg acgctcttcc gatctggaac gacgatgggc gtctcctg 58 <210> 37 <211> 43 <212> DNA <213> Artificial Sequence <220>

   <223> forward primer for secondary PCR <400> 37 aatgatacgg cgaccaccga gatctacact ctttccctac acg 43 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220>

   <223> reverse primer for secondary PCR;

   <400> 38 caagcagaag acggcata 18 <210> 39 <211> 1910 <212> DNA <213> Zea mays <220>

   <221> misc_feature <222> (1)..(1910) <223> ALS1-DNA sequence <400> 39 atggccaccg ccgccaccgc ggccgccgcg ctcaccggcg ccactaccgc tacgcccaag 60 tcgaggcgcc gagcccacca cttggccacc cggcgcgccc tcgccgcgcc catcaggtgc 120 tcagcgttgt cacgcgccac gccgacggct cccccggcca ctccgctacg tccgtggggc 180 cccaacgagc cccgcaaggg ctccgacatc ctcgtcgagg ctctcgagcg ctgtggcgtc 240 cgtgacgtct tcgcctaccc cggcggcgca tccatggaga tccaccaggc actcacccgc 300 tcccccgtca tcgccaacca cctcttccgc cacgaacaag gggaggcctt cgccgcctcc 360 ggctacgcgc gctcctcggg ccgcgttggc gtctgcatcg ccacctccgg ccccggcgcc 420 accaacctag tctctgcgct cgcagacgcg ttgctcgact ccgtccccat tgtcgccatc 480 acgggacagg tgccgcgacg catgattggc accgacgcct ttcaggagac gcccatcgtc 540 gaggtcaccc gctccatcac caagcacaac tacctggtcc tcgacgtcga cgacatcccc 600 cgcgtcgtgc aggaggcctt cttcctcgca tcctctggtc gcccggggcc ggtgcttgtt 660 gacatcccca aggacatcca gcagcagatg gcggtgccgg cctgggacac gcccatgagt 720 ctgcctgggt acatcgcgcg ccttcccaag cctcccgcga ctgaatttct tgagcaggtg 780 ctgcgtcttg ttggtgaatc acggcgccct gttctttatg ttggcggtgg ctgtgcagca 840

   Page 10

   20161011_BB2533PCT_SeqLst.txt tcaggtgagg agttgtgccg ctttgtggag ttgactggaa tcccagtcac aactactctt 900 atgggccttg gcaacttccc cagcgacgac ccactgtcac tgcgcatgct tggtatgcat 960 ggcacagtgt atgcaaatta tgcagtggat aaggccgatc tgttgcttgc atttggtgtg 1020 cggtttgatg atcgtgtgac agggaaaatt gaggcttttg caggcagagc taagattgtg 1080 cacattgata ttgatcctgc tgagattggc aagaacaagc agccacatgt gtccatctgt 1140 gcagatgtta agcttgcttt gcagggcatg aatactcttc tggaaggaag cacatcaaag 1200 aagagctttg acttcggctc atggcatgat gaattggatc agcaaaagcg ggagtttccc 1260 cttgggtata aaatcttcaa tgaggaaatc cagccacaat atgctattca ggttcttgat 1320 gagttgacga aggggaaggc catcattgcc acaggtgttg ggcagcacca gatgtgggcg 1380 gcacagtatt acacttacaa gcggccaagg cagtggctgt cttcagctgg tcttggggct 1440 atgggatttg gtttgccggc tgctgctggt gctgctgtgg ccaacccagg tgtcactgtt 1500 gttgacatcg acggagatgg tagcttcctc atgaacattc aggagctagc tatgatccgt 1560 attgagaacc tcccagtcaa ggtctttgtg ctaaacaacc agcacctcgg gatggtggtg 1620 cagtgggagg acaggttcta taaggccaat agagcacaca cattcttggg aaacccagag 1680 aacgaaagtg agatatatcc agattttgtg gcaattgcca aagggttcaa cattccagca 1740 gtccgtgtga caaagaagag cgaagtccat gcagcaatca agaagatgct tgaggctcca 1800 gggccgtacc tcttggatat aatcgtcccg caccaggagc atgtgttgcc tatgatccct 1860 agtggtgggg ctttcaagga tatgatcctg gatggtgatg gcaggactgt 1910 <210> 40 <211> 1910 <212> DNA <213> Zea mays <220>

   <221> misc_feature <222> (1)..(1910) <223> ALS2-DNA sequence <400> 40 atggccaccg ccgccgccgc gtctaccgcg ctcactggcg ccactaccgc tgcgcccaag 60 gcgaggcgcc gggcgcacct cctggccacc cgccgcgccc tcgccgcgcc catcaggtgc 120 tcagcggcgt cacccgccat gccgatggct cccccggcca ccccgctccg gccgtggggc 180 cccaccgatc cccgcaaggg cgccgacatc ctcgtcgagt ccctcgagcg ctgcggcgtc 240 cgcgacgtct tcgcctaccc cggcggcgcg tccatggaga tccaccaggc actcacccgc 300 tcccccgtca tcgccaacca cctcttccgc cacgagcaag gggaggcctt tgcggcctcc 360 ggctacgcgc gctcctcggg ccgcgtcggc gtctgcatcg ccacctccgg ccccggcgcc 420 accaaccttg tctccgcgct cgccgacgcg ctgctcgatt ccgtccccat ggtcgccatc 480 acgggacagg tgccgcgacg catgattggc accgacgcct tccaggagac gcccatcgtc 540 gaggtcaccc gctccatcac caagcacaac tacctggtcc tcgacgtcga cgacatcccc 600 cgcgtcgtgc aggaggcttt cttcctcgcc tcctctggtc gaccggggcc ggtgcttgtc 660 gacatcccca aggacatcca gcagcagatg gcggtgcctg tctgggacaa gcccatgagt 720 ctgcctgggt acattgcgcg ccttcccaag ccccctgcga ctgagttgct tgagcaggtg 780 ctgcgtcttg ttggtgaatc ccggcgccct gttctttatg ttggcggtgg ctgcgcagca 840 tctggtgagg agttgcgacg ctttgtggag ctgactggaa tcccggtcac aactactctt 900 atgggcctcg gcaacttccc cagcgacgac ccactgtctc tgcgcatgct aggtatgcat 960 ggcacggtgt atgcaaatta tgcagtggat aaggccgatc tgttgcttgc acttggtgtg 1020 cggtttgatg atcgtgtgac agggaagatt gaggcttttg caagcagggc taagattgtg 1080 cacgttgata ttgatccggc tgagattggc aagaacaagc agccacatgt gtccatctgt 1140 gcagatgtta agcttgcttt gcagggcatg aatgctcttc ttgaaggaag cacatcaaag 1200 aagagctttg actttggctc atggaacgat gagttggatc agcagaagag ggaattcccc 1260 cttgggtata aaacatctaa tgaggagatc cagccacaat atgctattca ggttcttgat 1320 gagctgacga aaggcgaggc catcatcggc acaggtgttg ggcagcacca gatgtgggcg 1380 gcacagtact acacttacaa gcggccaagg cagtggttgt cttcagctgg tcttggggct 1440 atgggatttg gtttgccggc tgctgctggt gcttctgtgg ccaacccagg tgttactgtt 1500 gttgacatcg atggagatgg tagctttctc atgaacgttc aggagctagc tatgatccga 1560 attgagaacc tcccggtgaa ggtctttgtg ctaaacaacc agcacctggg gatggtggtg 1620 cagtgggagg acaggttcta taaggccaac agagcgcaca catacttggg aaacccagag 1680 aatgaaagtg agatatatcc agatttcgtg acgatcgcca aagggttcaa cattccagcg 1740 gtccgtgtga caaagaagaa cgaagtccgc gcagcgataa agaagatgct cgagactcca 1800 gggccgtacc tcttggatat aatcgtccca caccaggagc atgtgttgcc tatgatccct 1860 agtggtgggg ctttcaagga tatgatcctg gatggtgatg gcaggactgt 1910 <210> 41 <211> 638 <212> PRT <213> Zea mays

   Page 11

   20161011_BB2533PCT_SeqLst.txt <220>

   <221> MISC_FEATURE <222> (1)..(638) <223> full length Zm-ALS2 protein <400> 41

   Met Ala Thr Ala Ala Ala Ala Ser Thr Ala Leu Thr Gly Ala Thr Thr 1 5 10 15 Ala Ala Pro Lys Ala Arg Arg Arg Ala His Leu Leu Ala Thr Arg Arg 20 25 30 Ala Leu Ala Ala Pro Ile Arg Cys Ser Ala Ala Ser Pro Ala Met Pro 35 40 45 Met Ala Pro Pro Ala Thr Pro Leu Arg Pro Trp Gly Pro Thr Glu Pro 50 55 60 Arg Lys Gly Ala Asp Ile Leu Val Glu Ser Leu Glu Arg Cys Gly Val 65 70 75 80 Arg Asp Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gln 85 90 95 Ala Leu Thr Arg Ser Pro Val Ile Ala Asn His Leu Phe Arg His Glu 100 105 110 Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr Ala Arg Ser Ser Gly Arg 115 120 125 Val Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 130 135 140 Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Met Val Ala Ile 145 150 155 160 Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu 165 170 175 Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu 180 185 190 Val Leu Asp Val Asp Asp Ile Pro Arg Val Val Gln Glu Ala Phe Phe 195 200 205 Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu Val Asp Ile Pro Lys 210 215 220 Asp Ile Gln Gln Gln Met Ala Val Pro Val Trp Asp Lys Pro Met Ser 225 230 235 240 Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys Pro Pro Ala Thr Glu Leu 245 250 255 Leu Glu Gln Val Leu Arg Leu Val Gly Glu Ser Arg Arg Pro Val Leu 260 265 270 Tyr Val Gly Gly Gly Cys Ala Ala Ser Gly Glu Glu Leu Arg Arg Phe 275 280 285 Val Glu Leu Thr Gly Ile Pro Val Thr Thr Thr Leu Met Gly Leu Gly 290 295 300 Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu Arg Met Leu Gly Met His 305 310 315 320 Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu 325 330 335 Ala Leu Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Ile Glu Ala 340 345 350 Phe Ala Ser Arg Ala Lys Ile Val His Val Asp Ile Asp Pro Ala Glu 355 360 365 Ile Gly Lys Asn Lys Gln Pro His Val Ser Ile Cys Ala Asp Val Lys 370 375 380 Leu Ala Leu Gln Gly Met Asn Ala Leu Leu Glu Gly Ser Thr Ser Lys 385 390 395 400 Lys Ser Phe Asp Phe Gly Ser Trp Asn Asp Glu Leu Asp Gln Gln Lys 405 410 415 Arg Glu Phe Pro Leu Gly Tyr Lys Thr Ser Asn Glu Glu Ile Gln Pro 420 425 430 Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr Lys Gly Glu Ala Ile 435 440 445 Ile Gly Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Tyr Tyr 450 455 460 Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser Ser Ala Gly Leu Gly Ala 465 470 475 480 Met Gly Phe Gly Leu Pro Ala Ala Ala Gly Ala Ser Val Ala Asn Pro 485 490 495

   Page 12

   20 1610 11_B B253 3PCT _Seq Lst. txt Gly Val Thr Val Val Asp Ile Asp Gly Asp Gly Ser Phe Leu Met Asn 500 505 510 Val Gln Glu Leu Ala Met Ile Arg Ile Glu Asn Leu Pro Val Lys Val 515 520 525 Phe Val Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp Glu Asp 530 535 540 Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Glu 545 550 555 560 Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val Thr Ile Ala Lys Gly Phe 565 570 575 Asn Ile Pro Ala Val Arg Val Thr Lys Lys Asn Glu Val Arg Ala Ala 580 585 590 Ile Lys Lys Met Leu Glu Thr Pro Gly Pro Tyr Leu Leu Asp Ile Ile 595 600 605 Val Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Ala 610 615 620 Phe Lys Asp Met Ile Leu Asp Gly Asp Gly Arg Thr Val Tyr 625 630 635 <210> 42 <211> 23 <212> DNA <213> Zea mays <400> 42

   gctgctcgat tccgtcccca tgg <210> 43 <211> 794 <212> DNA <213> Artificial sequence <220>

   <223> 794 bp polynucleotide modification template <400> 43 ttctgctcaa gcaactcagt cgcagggggc ttgggaaggc gcgcaatgta cccaggcaga ctcatgggct tgtcccagac aggcaccgcc atctgctgct ggatgtcctt ggggatgtcg acaagcaccg gccctggtcg accagaggag gcgaggaaga aagcctcctg cacgacgcgg gggatgtcgt cgacgtcgag gaccaggtag ttgtgcttgg tgatggagcg ggtgacctcg acgatgggcg tctcctggaa ggcgtcggtg ccaatcatgc gtcgcgacac ttggccggta atcgccacca tggggacgga atcgagcagc gcgtcggcga gcgcggagac aaggttggtg gcgccggggc cggaggtggc gatgcagacg ccgacgcggc ccgaggagcg cgcgtagccg gaggccgcaa aggcctcccc ttgctcgtgg cggaagaggt ggttggcgat gacgggggag cgggtgagtg cctggtggat ctccatggac gcgccgccgg ggtaggcgaa gacgtcgcgg acgccgcagc gctcgaggga ctcgacgagg atgtcggcgc ccttgcgggg atcggtgggg ccccacggcc ggagcggggt ggccggggga gccatcggca tggcgggtga cgccgctgag cacctgatgg gcgcggcgag ggcgcggcgg gtggccagga ggtgcgcccg gcgcctcgcc ttgggcgcag cggtagtggc gccagtgagc gcggtagacg cggcggcggc ggtggccatg gttgcggcgg ctgt <210> 44 <211> 127 <212> DNA <213> Artificial sequence <220>

   <223> 127 bp polynucleotide modification template oligo -1 <400> 44 aaccttgtct ccgcgctcgc cgacgcgttg ctcgactccg tccccattgt cgccatcacg ggacaggtgt cgcgacgcat gattggcacc gacgccttcc aggagacgcc catcgtcgag gtcaccc <210> 45 <211> 127 <212> DNA <213> artificial sequence

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   Page 13

   20161011_BB2533PCT_SeqLst.txt <220>

   <223> 127 bp polynucleotide modification template oligo-2 <400> 45 aaccttgtct ccgcgctcgc cgacgcgttg ctggactccg tgccgatggt cgccatcacg ggacaggtgt cccgacgcat gattggcacc gacgccttcc aggagacgcc catcgtcgag gtcaccc <210> 46 <211> 56493 <212> DNA <213> Artificial sequence <220>

   <223> Agrobacterium vector containing maize codon optimized Cas9 and

   120

   127 maize UBI promoter <400> 46 gtcggatcac cggaaaggac ccgtaaagtg gcgtggaggc catcaaacca cgtcaaataa atctgcatca acttaacgta aaaacaactt ggggcaacct catgtccccc cccccccccc gtttggtatg gcttcattca gctccggttc catgttgtgc aaaaaagcgg ttagctcctt ggccgcagtg ttatcactca tggttatggc atccgtaaga tgcttttctg tgactggtga tatgcggcga ccgagttgct cttgcccggc cagaacttta aaagtgctca tcattggaaa cttaccgctg ttgagatcca gttcgatgta atcttttact ttcaccagcg tttctgggtg aaagggaata agggcgacac ggaaatgttg ttgaagcatt tatcagggtt attgtctcat aaataaacaa ataggggttc cgcgcacatt aaccattatt atcatgacat taacctataa tcaagaattc ggagcttttg ccattctcac cacttgataa ccttattttt gacgagggga tcggaatcgc agaccgatac caggatcttg ctccttcatt acagaaacgg ctttttcaaa aattgcagtt tcatttgatg ctcgatgagt aacactggca gagcattacg ctgacttgac ttttgctgag ttgaaggatc agatcacgca aaagcaaaag ttcaaaatca ccaactggtc ctccctcact ttctggctgg atgatggggc ttcttcacga ggcagacctc agcgccagaa ggcttggacg ctagggcagg gcatgaaaaa gcggtggaaa gggggagggg atgttgtcta tccggcagcg gtcctgatca atcgtcaccc agcctccttt tcgccaatcc atcgacaatc ggaccggctt cgtcgaaggc gtctatcgcg tcaacggtgc cgccgcgctc gccggcatcg ccaacagtga agtagctgat tgtcatcagc gcctcgcaga ggaagcgaag ctgcgcgtcg gtgccggcat ggatgcgcgc gccatcgcgg gcattcccga tcagaaatga gcgccagtcg ttctccgcca gcatggcttc ggccagtgcg cagtaaagcg ccggctgctg aacccccaac tctacgccga cctcgttcaa caggtccagg tttgtcatgc ttgacacttt atcactgata taaagaatcc gcgcgttcaa tcggaccagc ccaacatacc cctgatcgta attctgagca tgattatgcc ggtgctgccg ggcctcctgc cccactatgg cattctgctg gcgctgtatg tgggcgcgct gtcggatcgt ttcgggcggc ccactgtcga ctacgccatc atggcgacag ggatcgtggc cggcatcacc ggggcgactg tcactgatgg cgatgagcgc gcgcggcact ggatggtcgc gggacctgtg ctcggtgggc ataatgatta tcatctacat atcacaacgt tcaattatga cgcaggtatc gtattaattg cagacaatac aaatcagcga cactgaatac ccctgcaggc atcgtggtgt cacgctcgtc ccaacgatca aggcgagtta catgatcccc cggtcctccg atcgttgtca gaagtaagtt agcactgcat aattctctta ctgtcatgcc gtactcaacc aagtcattct gagaatagtg gtcaacacgg gataataccg cgccacatag acgttcttcg gggcgaaaac tctcaaggat acccactcgt gcacccaact gatcttcagc agcaaaaaca ggaaggcaaa atgccgcaaa aatactcata ctcttccttt ttcaatatta gagcggatac atatttgaat gtatttagaa tccccgaaaa gtgccacctg acgtctaaga aaataggcgt atcacgaggc cctttcgtct cggattcagt cgtcactcat ggtgatttct aattaatagg ttgtattgat gttggacgag ccatcctatg gaactgcctc ggtgagtttt aatatggtat tgataatcct gatatgaata ttttctaatc agaattggtt aattggttgt gggacggcgg ctttgttgaa taaatcgaac tcttcccgac aacgcagacc gttccgtggc cacctacaac aaagctctca tcaaccgtgg gattcaggcc tggtatgagt cagcaacacc ggccgccaga gaggccgagc gcggccgtga gcccgtagcg ggctgctacg ggcgtctgac catggctctg ctgtagtgag tgggttgcgc tttctcggtc cttcaacgtt cctgacaacg accgcgagtc cctgctcgaa cgctgcgtcc gcccgcaaca gcggcgagag cggagcctgt ctgtcgccgg cctgctcctc aagcacggcc gcattgacgg cgtccccggc cgaaaaaccc gccgtttcca tctgcggtgc gcccggtcgc taggcgagca gcgcctgcct gaagctgcgg tcgtcggctc tcggcaccga atgcgtatga tcgagcagcg cccgcttgtt cctgaagtgc cgttccgcca gtttgcgtgt cgtcagaccg gcggcacgga tcactgtatt cggctgcaac aacataatat gtccaccaac ttatcagtga ggaggctggt ccggaggcca gacgtgaaac ctgtcgcgct cgacgctgtc ggcatcggcc gcgatctggt tcactcgaac gacgtcaccg cgttggtgca atttgcctgc gcacctgtgc ggccaatctt gctcgtctcg ctggccggcg cgcctttcct ttgggttctc tatatcgggc gggcggtagc cggcgcttat attgccgata tcggcttcat gagcgcctgt ttcgggttcg tgatgggcgg tttctccccc cacgctccgt

   Page 14

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   2940

   20161011_BB2533PCT_SeqLst.txt tcttcgccgc ggcagccttg aacggcctca atttcctgac gggctgtttc cttttgccgg 3000 agtcgcacaa aggcgaacgc cggccgttac gccgggaggc tctcaacccg ctcgcttcgt 3060 tccggtgggc ccggggcatg accgtcgtcg ccgccctgat ggcggtcttc ttcatcatgc 3120 aacttgtcgg acaggtgccg gccgcgcttt gggtcatttt cggcgaggat cgctttcact 3180 gggacgcgac cacgatcggc atttcgcttg ccgcatttgg cattctgcat tcactcgccc 3240 aggcaatgat caccggccct gtagccgccc ggctcggcga aaggcgggca ctcatgctcg 3300 gaatgattgc cgacggcaca ggctacatcc tgcttgcctt cgcgacacgg ggatggatgg 3360 cgttcccgat catggtcctg cttgcttcgg gtggcatcgg aatgccggcg ctgcaagcaa 3420 tgttgtccag gcaggtggat gaggaacgtc aggggcagct gcaaggctca ctggcggcgc 3480 tcaccagcct gacctcgatc gtcggacccc tcctcttcac ggcgatctat gcggcttcta 3540 taacaacgtg gaacgggtgg gcatggattg caggcgctgc cctctacttg ctctgcctgc 3600 cggcgctgcg tcgcgggctt tggagcggcg cagggcaacg agccgatcgc tgatcgtgga 3660 aacgataggc ctatgccatg cgggtcaagg cgacttccgg caagctatac gcgccctagg 3720 agtgcggttg gaacgttggc ccagccagat actcccgatc acgagcagga cgccgatgat 3780 ttgaagcgca ctcagcgtct gatccaagaa caaccatcct agcaacacgg cggtccccgg 3840 gctgagaaag cccagtaagg aaacaactgt aggttcgagt cgcgagatcc cccggaacca 3900 aaggaagtag gttaaacccg ctccgatcag gccgagccac gccaggccga gaacattggt 3960 tcctgtaggc atcgggattg gcggatcaaa cactaaagct actggaacga gcagaagtcc 4020 tccggccgcc agttgccagg cggtaaaggt gagcagaggc acgggaggtt gccacttgcg 4080 ggtcagcacg gttccgaacg ccatggaaac cgcccccgcc aggcccgctg cgacgccgac 4140 aggatctagc gctgcgtttg gtgtcaacac caacagcgcc acgcccgcag ttccgcaaat 4200 agcccccagg accgccatca atcgtatcgg gctacctagc agagcggcag agatgaacac 4260 gaccatcagc ggctgcacag cgcctaccgt cgccgcgacc ccgcccggca ggcggtagac 4320 cgaaataaac aacaagctcc agaatagcga aatattaagt gcgccgagga tgaagatgcg 4380 catccaccag attcccgttg gaatctgtcg gacgatcatc acgagcaata aacccgccgg 4440 caacgcccgc agcagcatac cggcgacccc tcggcctcgc tgttcgggct ccacgaaaac 4500 gccggacaga tgcgccttgt gagcgtcctt ggggccgtcc tcctgtttga agaccgacag 4560 cccaatgatc tcgccgtcga tgtaggcgcc gaatgccacg gcatctcgca accgttcagc 4620 gaacgcctcc atgggctttt tctcctcgtg ctcgtaaacg gacccgaaca tctctggagc 4680 tttcttcagg gccgacaatc ggatctcgcg gaaatcctgc acgtcggccg ctccaagccg 4740 tcgaatctga gccttaatca caattgtcaa ttttaatcct ctgtttatcg gcagttcgta 4800 gagcgcgccg tgcgtcccga gcgatactga gcgaagcaag tgcgtcgagc agtgcccgct 4860 tgttcctgaa atgccagtaa agcgctggct gctgaacccc cagccggaac tgaccccaca 4920 aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc caccaggccg 4980 ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt cacgcgggtg 5040 gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc 5100 gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat 5160 atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc gatcaggacc 5220 tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag cagcgacacc 5280 gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac 5340 aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag gtcctggcaa 5400 agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc gtactccaac 5460 acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc 5520 ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc 5580 ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg 5640 tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg 5700 tgtttcagca acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg 5760 gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga cttcgccaaa 5820 cctgccgcct cctgttcgag acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg 5880 gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc 5940 gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt 6000 tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc cttccggtca 6060 aacgtccgat tcattcaccc tccttgcggg attgccccga ctcacgccgg ggcaatgtgc 6120 ccttattcct gatttgaccc gcctggtgcc ttggtgtcca gataatccac cttatcggca 6180 atgaagtcgg tcccgtagac cgtctggccg tccttctcgt acttggtatt ccgaatcttg 6240 ccctgcacga ataccagcga ccccttgccc aaatacttgc cgtgggcctc ggcctgagag 6300 ccaaaacact tgatgcggaa gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc 6360 cactcttcat taaccgctat atcgaaaatt gcttgcggct tgttagaatt gccatgacgt 6420 acctcggtgt cacgggtaag attaccgata aactggaact gattatggct catatcgaaa 6480 gtctccttga gaaaggagac tctagtttag ctaaacattg gttccgctgt caagaacttt 6540 agcggctaaa attttgcggg ccgcgaccaa aggtgcgagg ggcggcttcc gctgtgtaca 6600 accagatatt tttcaccaac atccttcgtc tgctcgatga gcggggcatg acgaaacatg 6660 agctgtcgga gagggcaggg gtttcaattt cgtttttatc agacttaacc aacggtaagg 6720 ccaacccctc gttgaaggtg atggaggcca ttgccgacgc cctggaaact cccctacctc 6780 ttctcctgga gtccaccgac cttgaccgcg aggcactcgc ggagattgcg ggtcatcctt 6840 tcaagagcag cgtgccgccc ggatacgaac gcatcagtgt ggttttgccg tcacataagg 6900 cgtttatcgt aaagaaatgg ggcgacgaca cccgaaaaaa gctgcgtgga aggctctgac 6960 gccaagggtt agggcttgca cttccttctt tagccgctaa aacggcccct tctctgcggg 7020

   Page 15

   20161011_BB2533PCT_SeqLst.txt ccgtcggctc gcgcatcata tcgacatcct caacggaagc cgtgccgcga atggcatcgg 7080 gcgggtgcgc tttgacagtt gttttctatc agaaccccta cgtcgtgcgg ttcgattagc 7140 tgtttgtctt gcaggctaaa cactttcggt atatcgtttg cctgtgcgat aatgttgcta 7200 atgatttgtt gcgtaggggt tactgaaaag tgagcgggaa agaagagttt cagaccatca 7260 aggagcgggc caagcgcaag ctggaacgcg acatgggtgc ggacctgttg gccgcgctca 7320 acgacccgaa aaccgttgaa gtcatgctca acgcggacgg caaggtgtgg cacgaacgcc 7380 ttggcgagcc gatgcggtac atctgcgaca tgcggcccag ccagtcgcag gcgattatag 7440 aaacggtggc cggattccac ggcaaagagg tcacgcggca ttcgcccatc ctggaaggcg 7500 agttcccctt ggatggcagc cgctttgccg gccaattgcc gccggtcgtg gccgcgccaa 7560 cctttgcgat ccgcaagcgc gcggtcgcca tcttcacgct ggaacagtac gtcgaggcgg 7620 gcatcatgac ccgcgagcaa tacgaggtca ttaaaagcgc cgtcgcggcg catcgaaaca 7680 tcctcgtcat tggcggtact ggctcgggca agaccacgct cgtcaacgcg atcatcaatg 7740 aaatggtcgc cttcaacccg tctgagcgcg tcgtcatcat cgaggacacc ggcgaaatcc 7800 agtgcgccgc agagaacgcc gtccaatacc acaccagcat cgacgtctcg atgacgctgc 7860 tgctcaagac aacgctgcgt atgcgccccg accgcatcct ggtcggtgag gtacgtggcc 7920 ccgaagccct tgatctgttg atggcctgga acaccgggca tgaaggaggt gccgccaccc 7980 tgcacgcaaa caaccccaaa gcgggcctga gccggctcgc catgcttatc agcatgcacc 8040 cggattcacc gaaacccatt gagccgctga ttggcgaggc ggttcatgtg gtcgtccata 8100 tcgccaggac ccctagcggc cgtcgagtgc aagaaattct cgaagttctt ggttacgaga 8160 acggccagta catcaccaaa accctgtaag gagtatttcc aatgacaacg gctgttccgt 8220 tccgtctgac catgaatcgc ggcattttgt tctaccttgc cgtgttcttc gttctcgctc 8280 tcgcgttatc cgcgcatccg gcgatggcct cggaaggcac cggcggcagc ttgccatatg 8340 agagctggct gacgaacctg cgcaactccg taaccggccc ggtggccttc gcgctgtcca 8400 tcatcggcat cgtcgtcgcc ggcggcgtgc tgatcttcgg cggcgaactc aacgccttct 8460 tccgaaccct gatcttcctg gttctggtga tggcgctgct ggtcggcgcg cagaacgtga 8520 tgagcacctt cttcggtcgt ggtgccgaaa tcgcggccct cggcaacggg gcgctgcacc 8580 aggtgcaagt cgcggcggcg gatgccgtgc gtgcggtagc ggctggacgg ctcgcctaat 8640 catggctctg cgcacgatcc ccatccgtcg cgcaggcaac cgagaaaacc tgttcatggg 8700 tggtgatcgt gaactggtga tgttctcggg cctgatggcg tttgcgctga ttttcagcgc 8760 ccaagagctg cgggccaccg tggtcggtct gatcctgtgg ttcggggcgc tctatgcgtt 8820 ccgaatcatg gcgaaggccg atccgaagat gcggttcgtg tacctgcgtc accgccggta 8880 caagccgtat tacccggccc gctcgacccc gttccgcgag aacaccaata gccaagggaa 8940 gcaataccga tgatccaagc aattgcgatt gcaatcgcgg gcctcggcgc gcttctgttg 9000 ttcatcctct ttgcccgcat ccgcgcggtc gatgccgaac tgaaactgaa aaagcatcgt 9060 tccaaggacg ccggcctggc cgatctgctc aactacgccg ctgtcgtcga tgacggcgta 9120 atcgtgggca agaacggcag ctttatggct gcctggctgt acaagggcga tgacaacgca 9180 agcagcaccg accagcagcg cgaagtagtg tccgcccgca tcaaccaggc cctcgcgggc 9240 ctgggaagtg ggtggatgat ccatgtggac gccgtgcggc gtcctgctcc gaactacgcg 9300 gagcggggcc tgtcggcgtt ccctgaccgt ctgacggcag cgattgaaga agagcgctcg 9360 gtcttgcctt gctcgtcggt gatgtacttc accagctccg cgaagtcgct cttcttgatg 9420 gagcgcatgg ggacgtgctt ggcaatcacg cgcacccccc ggccgtttta gcggctaaaa 9480 aagtcatggc tctgccctcg ggcggaccac gcccatcatg accttgccaa gctcgtcctg 9540 cttctcttcg atcttcgcca gcagggcgag gatcgtggca tcaccgaacc gcgccgtgcg 9600 cgggtcgtcg gtgagccaga gtttcagcag gccgcccagg cggcccaggt cgccattgat 9660 gcgggccagc tcgcggacgt gctcatagtc cacgacgccc gtgattttgt agccctggcc 9720 gacggccagc aggtaggccg acaggctcat gccggccgcc gccgcctttt cctcaatcgc 9780 tcttcgttcg tctggaaggc agtacacctt gataggtggg ctgcccttcc tggttggctt 9840 ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg gccagcctcg 9900 cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga agaaggaaca 9960 cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt ggatacacca 10020 aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata 10080 taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa agcgctgctt 10140 ccctgctgtt ttgtggaata tctaccgact ggaaacaggc aaatgcagga aattactgaa 10200 ctgaggggac aggcgagaga cgatgccaaa gagctacacc gacgagctgg ccgagtgggt 10260 tgaatcccgc gcggccaaga agcgccggcg tgatgaggct gcggttgcgt tcctggcggt 10320 gagggcggat gtcgaggcgg cgttagcgtc cggctatgcg ctcgtcacca tttgggagca 10380 catgcgggaa acggggaagg tcaagttctc ctacgagacg ttccgctcgc acgccaggcg 10440 gcacatcaag gccaagcccg ccgatgtgcc cgcaccgcag gccaaggctg cggaacccgc 10500 gccggcaccc aagacgccgg agccacggcg gccgaagcag gggggcaagg ctgaaaagcc 10560 ggcccccgct gcggccccga ccggcttcac cttcaaccca acaccggaca aaaaggatct 10620 actgtaatgg cgaaaattca catggttttg cagggcaagg gcggggtcgg caagtcggcc 10680 atcgccgcga tcattgcgca gtacaagatg gacaaggggc agacaccctt gtgcatcgac 10740 accgacccgg tgaacgcgac gttcgagggc tacaaggccc tgaacgtccg ccggctgaac 10800 atcatggccg gcgacgaaat taactcgcgc aacttcgaca ccctggtcga gctgattgcg 10860 ccgaccaagg atgacgtggt gatcgacaac ggtgccagct cgttcgtgcc tctgtcgcat 10920 tacctcatca gcaaccaggt gccggctctg ctgcaagaaa tggggcatga gctggtcatc 10980 cataccgtcg tcaccggcgg ccaggctctc ctggacacgg tgagcggctt cgcccagctc 11040 gccagccagt tcccggccga agcgcttttc gtggtctggc tgaacccgta ttgggggcct 11100

   Page 16

   20161011_BB2533PCT_SeqLst.txt atcgagcatg agggcaagag ctttgagcag atgaaggcgt acacggccaa caaggcccgc 11160 gtgtcgtcca tcatccagat tccggccctc aaggaagaaa cctacggccg cgatttcagc 11220 gacatgctgc aagagcggct gacgttcgac caggcgctgg ccgatgaatc gctcacgatc 11280 atgacgcggc aacgcctcaa gatcgtgcgg cgcggcctgt ttgaacagct cgacgcggcg 11340 gccgtgctat gagcgaccag attgaagagc tgatccggga gattgcggcc aagcacggca 11400 tcgccgtcgg ccgcgacgac ccggtgctga tcctgcatac catcaacgcc cggctcatgg 11460 ccgacagtgc ggccaagcaa gaggaaatcc ttgccgcgtt caaggaagag ctggaaggga 11520 tcgcccatcg ttggggcgag gacgccaagg ccaaagcgga gcggatgctg aacgcggccc 11580 tggcggccag caaggacgca atggcgaagg taatgaagga cagcgccgcg caggcggccg 11640 aagcgatccg cagggaaatc gacgacggcc ttggccgcca gctcgcggcc aaggtcgcgg 11700 acgcgcggcg cgtggcgatg atgaacatga tcgccggcgg catggtgttg ttcgcggccg 11760 ccctggtggt gtgggcctcg ttatgaatcg cagaggcgca gatgaaaaag cccggcgttg 11820 ccgggctttg tttttgcgtt agctgggctt gtttgacagg cccaagctct gactgcgccc 11880 gcgctcgcgc tcctgggcct gtttcttctc ctgctcctgc ttgcgcatca gggcctggtg 11940 ccgtcgggct gcttcacgca tcgaatccca gtcgccggcc agctcgggat gctccgcgcg 12000 catcttgcgc gtcgccagtt cctcgatctt gggcgcgtga atgcccatgc cttccttgat 12060 ttcgcgcacc atgtccagcc gcgtgtgcag ggtctgcaag cgggcttgct gttgggcctg 12120 ctgctgctgc caggcggcct ttgtacgcgg cagggacagc aagccggggg cattggactg 12180 tagctgctgc aaacgcgcct gctgacggtc tacgagctgt tctaggcggt cctcgatgcg 12240 ctccacctgg tcatgctttg cctgcacgta gagcgcaagg gtctgctggt aggtctgctc 12300 gatgggcgcg gattctaaga gggcctgctg ttccgtctcg gcctcctggg ccgcctgtag 12360 caaatcctcg ccgctgttgc cgctggactg ctttactgcc ggggactgct gttgccctgc 12420 tcgcgccgtc gtcgcagttc ggcttgcccc cactcgattg actgcttcat ttcgagccgc 12480 agcgatgcga tctcggattg cgtcaacgga cggggcagcg cggaggtgtc cggcttctcc 12540 ttgggtgagt cggtcgatgc catagccaaa ggtttccttc caaaatgcgt ccattgctgg 12600 accgtgtttc tcattgatgc ccgcaagcat cttcggcttg accgccaggt caagcgcgcc 12660 ttcatgggcg gtcatgacgg acgccgccat gaccttgccg ccgttgttct cgatgtagcc 12720 gcgtaatgag gcaatggtgc cgcccatcgt cagcgtgtca tcgacaacga tgtacttctg 12780 gccggggatc acctccccct cgaaagtcgg gttgaacgcc aggcgatgat ctgaaccggc 12840 tccggttcgg gcgaccttct cccgctgcac aatgtccgtt tcgacctcaa ggccaaggcg 12900 gtcggccaga acgaccgcca tcatggccgg aatcttgttg ttccccgccg cctcgacggc 12960 gaggactgga acgatgcggg gcttgtcgtc gccgatcagc gtcttgagct gggcaacagt 13020 gtcgtccgaa atcaggcgct cgaccaaatt aagcgccgct tccgcgtcgc cctgcttcgc 13080 agcctggtat tcaggctcgt tggtcaaaga accaaggtcg ccgttgcgaa ccaccttcgg 13140 gaagtctccc cacggtgcgc gctcggctct gctgtagctg ctcaagacgc ctcccttttt 13200 agccgctaaa actctaacga gtgcgcccgc gactcaactt gacgctttcg gcacttacct 13260 gtgccttgcc acttgcgtca taggtgatgc ttttcgcact cccgatttca ggtactttat 13320 cgaaatctga ccgggcgtgc attacaaagt tcttccccac ctgttggtaa atgctgccgc 13380 tatctgcgtg gacgatgctg ccgtcgtggc gctgcgactt atcggccttt tgggccatat 13440 agatgttgta aatgccaggt ttcagggccc cggctttatc taccttctgg ttcgtccatg 13500 cgccttggtt ctcggtctgg acaattcttt gcccattcat gaccaggagg cggtgtttca 13560 ttgggtgact cctgacggtt gcctctggtg ttaaacgtgt cctggtcgct tgccggctaa 13620 aaaaaagccg acctcggcag ttcgaggccg gctttcccta gagccgggcg cgtcaaggtt 13680 gttccatcta ttttagtgaa ctgcgttcga tttatcagtt actttcctcc cgctttgtgt 13740 ttcctcccac tcgtttccgc gtctagccga cccctcaaca tagcggcctc ttcttgggct 13800 gcctttgcct cttgccgcgc ttcgtcacgc tcggcttgca ccgtcgtaaa gcgctcggcc 13860 tgcctggccg cctcttgcgc cgccaacttc ctttgctcct ggtgggcctc ggcgtcggcc 13920 tgcgccttcg ctttcaccgc tgccaactcc gtgcgcaaac tctccgcttc gcgcctggtg 13980 gcgtcgcgct cgccgcgaag cgcctgcatt tcctggttgg ccgcgtccag ggtcttgcgg 14040 ctctcttctt tgaatgcgcg ggcgtcctgg tgagcgtagt ccagctcggc gcgcagctcc 14100 tgcgctcgac gctccacctc gtcggcccgc tgcgtcgcca gcgcggcccg ctgctcggct 14160 cctgccaggg cggtgcgtgc ttcggccagg gcttgccgct ggcgtgcggc cagctcggcc 14220 gcctcggcgg cctgctgctc tagcaatgta acgcgcgcct gggcttcttc cagctcgcgg 14280 gcctgcgcct cgaaggcgtc ggccagctcc ccgcgcacgg cttccaactc gttgcgctca 14340 cgatcccagc cggcttgcgc tgcctgcaac gattcattgg caagggcctg ggcggcttgc 14400 cagagggcgg ccacggcctg gttgccggcc tgctgcaccg cgtccggcac ctggactgcc 14460 agcggggcgg cctgcgccgt gcgctggcgt cgccattcgc gcatgccggc gctggcgtcg 14520 ttcatgttga cgcgggcggc cttacgcact gcatccacgg tcgggaagtt ctcccggtcg 14580 ccttgctcga acagctcgtc cgcagccgca aaaatgcggt cgcgcgtctc tttgttcagt 14640 tccatgttgg ctccggtaat tggtaagaat aataatactc ttacctacct tatcagcgca 14700 agagtttagc tgaacagttc tcgacttaac ggcaggtttt ttagcggctg aagggcaggc 14760 aaaaaaagcc ccgcacggtc ggcgggggca aagggtcagc gggaagggga ttagcgggcg 14820 tcgggcttct tcatgcgtcg gggccgcgct tcttgggatg gagcacgacg aagcgcgcac 14880 gcgcatcgtc ctcggcccta tcggcccgcg tcgcggtcag gaacttgtcg cgcgctaggt 14940 cctccctggt gggcaccagg ggcatgaact cggcctgctc gatgtaggtc cactccatga 15000 ccgcatcgca gtcgaggccg cgttccttca ccgtctcttg caggtcgcgg tacgcccgct 15060 cgttgagcgg ctggtaacgg gccaattggt cgtaaatggc tgtcggccat gagcggcctt 15120 tcctgttgag ccagcagccg acgacgaagc cggcaatgca ggcccctggc acaaccaggc 15180

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   20161011_BB2533PCT_SeqLst.txt cgacgccggg ggcaggggat ggcagcagct cgccaaccag gaaccccgcc gcgatgatgc 15240 cgatgccggt caaccagccc ttgaaactat ccggccccga aacacccctg cgcattgcct 15300 ggatgctgcg ccggatagct tgcaacatca ggagccgttt cttttgttcg tcagtcatgg 15360 tccgccctca ccagttgttc gtatcggtgt cggacgaact gaaatcgcaa gagctgccgg 15420 tatcggtcca gccgctgtcc gtgtcgctgc tgccgaagca cggcgagggg tccgcgaacg 15480 ccgcagacgg cgtatccggc cgcagcgcat cgcccagcat ggccccggtc agcgagccgc 15540 cggccaggta gcccagcatg gtgctgttgg tcgccccggc caccagggcc gacgtgacga 15600 aatcgccgtc attccctctg gattgttcgc tgctcggcgg ggcagtgcgc cgcgccggcg 15660 gcgtcgtgga tggctcgggt tggctggcct gcgacggccg gcgaaaggtg cgcagcagct 15720 cgttatcgac cggctgcggc gtcggggccg ccgccttgcg ctgcggtcgg tgttccttct 15780 tcggctcgcg cagcttgaac agcatgatcg cggaaaccag cagcaacgcc gcgcctacgc 15840 ctcccgcgat gtagaacagc atcggattca ttcttcggtc ctccttgtag cggaaccgtt 15900 gtctgtgcgg cgcgggtggc ccgcgccgct gtctttgggg atcagccctc gatgagcgcg 15960 accagtttca cgtcggcaag gttcgcctcg aactcctggc cgtcgtcctc gtacttcaac 16020 caggcatagc cttccgccgg cggccgacgg ttgaggataa ggcgggcagg gcgctcgtcg 16080 tgctcgacct ggacgatggc ctttttcagc ttgtccgggt ccggctcctt cgcgcccttt 16140 tccttggcgt ccttaccgtc ctggtcgccg tcctcgccgt cctggccgtc gccggcctcc 16200 gcgtcacgct cggcatcagt ctggccgttg aaggcatcga cggtgttggg atcgcggccc 16260 ttctcgtcca ggaactcgcg cagcagcttg accgtgccgc gcgtgatttc ctgggtgtcg 16320 tcgtcaagcc acgcctcgac ttcctccggg cgcttcttga aggccgtcac cagctcgttc 16380 accacggtca cgtcgcgcac gcggccggtg ttgaacgcat cggcgatctt ctccggcagg 16440 tccagcagcg tgacgtgctg ggtgatgaac gccggcgact tgccgatttc cttggcgata 16500 tcgcctttct tcttgccctt cgccagctcg cggccaatga agtcggcaat ttcgcgcggg 16560 gtcagctcgt tgcgttgcag gttctcgata acctggtcgg cttcgttgta gtcgttgtcg 16620 atgaacgccg ggatggactt cttgccggcc cacttcgagc cacggtagcg gcgggcgccg 16680 tgattgatga tatagcggcc cggctgctcc tggttctcgc gcaccgaaat gggtgacttc 16740 accccgcgct ctttgatcgt ggcaccgatt tccgcgatgc tctccgggga aaagccgggg 16800 ttgtcggccg tccgcggctg atgcggatct tcgtcgatca ggtccaggtc cagctcgata 16860 gggccggaac cgccctgaga cgccgcagga gcgtccagga ggctcgacag gtcgccgatg 16920 ctatccaacc ccaggccgga cggctgcgcc gcgcctgcgg cttcctgagc ggccgcagcg 16980 gtgtttttct tggtggtctt ggcttgagcc gcagtcattg ggaaatctcc atcttcgtga 17040 acacgtaatc agccagggcg cgaacctctt tcgatgcctt gcgcgcggcc gttttcttga 17100 tcttccagac cggcacaccg gatgcgaggg catcggcgat gctgctgcgc aggccaacgg 17160 tggccggaat catcatcttg gggtacgcgg ccagcagctc ggcttggtgg cgcgcgtggc 17220 gcggattccg cgcatcgacc ttgctgggca ccatgccaag gaattgcagc ttggcgttct 17280 tctggcgcac gttcgcaatg gtcgtgacca tcttcttgat gccctggatg ctgtacgcct 17340 caagctcgat gggggacagc acatagtcgg ccgcgaagag ggcggccgcc aggccgacgc 17400 caagggtcgg ggccgtgtcg atcaggcaca cgtcgaagcc ttggttcgcc agggccttga 17460 tgttcgcccc gaacagctcg cgggcgtcgt ccagcgacag ccgttcggcg ttcgccagta 17520 ccgggttgga ctcgatgagg gcgaggcgcg cggcctggcc gtcgccggct gcgggtgcgg 17580 tttcggtcca gccgccggca gggacagcgc cgaacagctt gcttgcatgc aggccggtag 17640 caaagtcctt gagcgtgtag gacgcattgc cctgggggtc caggtcgatc acggcaaccc 17700 gcaagccgcg ctcgaaaaag tcgaaggcaa gatgcacaag ggtcgaagtc ttgccgacgc 17760 cgcctttctg gttggccgtg accaaagttt tcatcgtttg gtttcctgtt ttttcttggc 17820 gtccgcttcc cacttccgga cgatgtacgc ctgatgttcc ggcagaaccg ccgttacccg 17880 cgcgtacccc tcgggcaagt tcttgtcctc gaacgcggcc cacacgcgat gcaccgcttg 17940 cgacactgcg cccctggtca gtcccagcga cgttgcgaac gtcgcctgtg gcttcccatc 18000 gactaagacg ccccgcgcta tctcgatggt ctgctgcccc acttccagcc cctggatcgc 18060 ctcctggaac tggctttcgg taagccgttt cttcatggat aacacccata atttgctccg 18120 cgccttggtt gaacatagcg gtgacagccg ccagcacatg agagaagttt agctaaacat 18180 ttctcgcacg tcaacacctt tagccgctaa aactcgtcct tggcgtaaca aaacaaaagc 18240 ccggaaaccg ggctttcgtc tcttgccgct tatggctctg cacccggctc catcaccaac 18300 aggtcgcgca cgcgcttcac tcggttgcgg atcgacactg ccagcccaac aaagccggtt 18360 gccgccgccg ccaggatcgc gccgatgatg ccggccacac cggccatcgc ccaccaggtc 18420 gccgccttcc ggttccattc ctgctggtac tgcttcgcaa tgctggacct cggctcacca 18480 taggctgacc gctcgatggc gtatgccgct tctccccttg gcgtaaaacc cagcgccgca 18540 ggcggcattg ccatgctgcc cgccgctttc ccgaccacga cgcgcgcacc aggcttgcgg 18600 tccagacctt cggccacggc gagctgcgca aggacataat cagccgccga cttggctcca 18660 cgcgcctcga tcagctcttg cactcgcgcg aaatccttgg cctccacggc cgccatgaat 18720 cgcgcacgcg gcgaaggctc cgcagggccg gcgtcgtgat cgccgccgag aatgcccttc 18780 accaagttcg acgacacgaa aatcatgctg acggctatca ccatcatgca gacggatcgc 18840 acgaacccgc tgaattgaac acgagcacgg cacccgcgac cactatgcca agaatgccca 18900 aggtaaaaat tgccggcccc gccatgaagt ccgtgaatgc cccgacggcc gaagtgaagg 18960 gcaggccgcc acccaggccg ccgccctcac tgcccggcac ctggtcgctg aatgtcgatg 19020 ccagcacctg cggcacgtca atgcttccgg gcgtcgcgct cgggctgatc gcccatcccg 19080 ttactgcccc gatcccggca atggcaagga ctgccagcgc tgccattttt ggggtgaggc 19140 cgttcgcggc cgaggggcgc agcccctggg gggatgggag gcccgcgtta gcgggccggg 19200 agggttcgag aagggggggc accccccttc ggcgtgcgcg gtcacgcgca cagggcgcag 19260

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   20161011_BB2533PCT_SeqLst.txt ccctggttaa aaacaaggtt tataaatatt ggtttaaaag caggttaaaa gacaggttag 19320 cggtggccga aaaacgggcg gaaacccttg caaatgctgg attttctgcc tgtggacagc 19380 ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac tctgcccctc aagtgtcaag 19440 gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt gtcaataccg cagggcactt 19500 atccccaggc ttgtccacat catctgtggg aaactcgcgt aaaatcaggc gttttcgccg 19560 atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc gagcctgccc ctcatctgtc 19620 aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt ccgcccctca tctgtcagtg 19680 agggccaagt tttccgcgag gtatccacaa cgccggcggc cgcggtgtct cgcacacggc 19740 ttcgacggcg tttctggcgc gtttgcaggg ccatagacgg ccgccagccc agcggcgagg 19800 gcaaccagcc cggtgagcgt cggaaaggcg ctggaagccc cgtagcgacg cggagagggg 19860 cgagacaagc caagggcgca ggctcgatgc gcagcacgac atagccggtt ctcgcaagga 19920 cgagaatttc cctgcggtgc ccctcaagtg tcaatgaaag tttccaacgc gagccattcg 19980 cgagagcctt gagtccacgc tagatgagag ctttgttgta ggtggaccag ttggtgattt 20040 tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 20100 tcaactcagc aaaagttcga tttattcaac aaagccacgt tgtgtctcaa aatctctgat 20160 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa 20220 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tcgactctag 20280 agctcgttcc tcgaggcctc gaggcctcga ggaacggtac ctgcggggaa gcttacaata 20340 atgtgtgttg ttaagtcttg ttgcctgtca tcgtctgact gactttcgtc ataaatcccg 20400 gcctccgtaa cccagctttg ggcaagctca cggatttgat ccggcggaac gggaatatcg 20460 agatgccggg ctgaacgctg cagttccagc tttccctttc gggacaggta ctccagctga 20520 ttgattatct gctgaagggt cttggttcca cctcctggca caatgcgaat gattacttga 20580 gcgcgatcgg gcatccaatt ttctcccgtc aggtgcgtgg tcaagtgcta caaggcacct 20640 ttcagtaacg agcgaccgtc gatccgtcgc cgggatacgg acaaaatgga gcgcagtagt 20700 ccatcgaggg cggcgaaagc ctcgccaaaa gcaatacgtt catctcgcac agcctccaga 20760 tccgatcgag ggtcttcggc gtaggcagat agaagcatgg atacattgct tgagagtatt 20820 ccgatggact gaagtatggc ttccatcttt tctcgtgtgt ctgcatctat ttcgagaaag 20880 cccccgatgc ggcgcaccgc aacgcgaatt gccatactat ccgaaagtcc cagcaggcgc 20940 gcttgatagg aaaaggtttc atactcggcc gatcgcagac gggcactcac gaccttgaac 21000 ccttcaactt tcagggatcg atgctggttg atggtagtct cactcgacgt ggctctggtg 21060 tgttttgaca tagcttcctc caaagaaagc ggaaggtctg gatactccag cacgaaatgt 21120 gcccgggtag acggatggaa gtctagccct gctcaatatg aaatcaacag tacatttaca 21180 gtcaatactg aatatacttg ctacatttgc aattgtctta taacgaatgt gaaataaaaa 21240 tagtgtaaca acgcttttac tcatcgataa tcacaaaaac atttatacga acaaaaatac 21300 aaatgcactc cggtttcaca ggataggcgg gatcagaata tgcaactttt gacgttttgt 21360 tctttcaaag ggggtgctgg caaaaccacc gcactcatgg gcctttgcgc tgctttggca 21420 aatgacggta aacgagtggc cctctttgat gccgacgaaa accggcctct gacgcgatgg 21480 agagaaaacg ccttacaaag cagtactggg atcctcgctg tgaagtctat tccgccgacg 21540 aaatgcccct tcttgaagca gcctatgaaa atgccgagct cgaaggattt gattatgcgt 21600 tggccgatac gcgtggcggc tcgagcgagc tcaacaacac aatcatcgct agctcaaacc 21660 tgcttctgat ccccaccatg ctaacgccgc tcgacatcga tgaggcacta tctacctacc 21720 gctacgtcat cgagctgctg ttgagtgaaa atttggcaat tcctacagct gttttgcgcc 21780 aacgcgtccc ggtcggccga ttgacaacat cgcaacgcag gatgtcagag acgctagaga 21840 gccttccagt tgtaccgtct cccatgcatg aaagagatgc atttgccgcg atgaaagaac 21900 gcggcatgtt gcatcttaca ttactaaaca cgggaactga tccgacgatg cgcctcatag 21960 agaggaatct tcggattgcg atggaggaag tcgtggtcat ttcgaaactg atcagcaaaa 22020 tcttggaggc ttgaagatgg caattcgcaa gcccgcattg tcggtcggcg aagcacggcg 22080 gcttgctggt gctcgacccg agatccacca tcccaacccg acacttgttc cccagaagct 22140 ggacctccag cacttgcctg aaaaagccga cgagaaagac cagcaacgtg agcctctcgt 22200 cgccgatcac atttacagtc ccgatcgaca acttaagcta actgtggatg cccttagtcc 22260 acctccgtcc ccgaaaaagc tccaggtttt tctttcagcg cgaccgcccg cgcctcaagt 22320 gtcgaaaaca tatgacaacc tcgttcggca atacagtccc tcgaagtcgc tacaaatgat 22380 tttaaggcgc gcgttggacg atttcgaaag catgctggca gatggatcat ttcgcgtggc 22440 cccgaaaagt tatccgatcc cttcaactac agaaaaatcc gttctcgttc agacctcacg 22500 catgttcccg gttgcgttgc tcgaggtcgc tcgaagtcat tttgatccgt tggggttgga 22560 gaccgctcga gctttcggcc acaagctggc taccgccgcg ctcgcgtcat tctttgctgg 22620 agagaagcca tcgagcaatt ggtgaagagg gacctatcgg aacccctcac caaatattga 22680 gtgtaggttt gaggccgctg gccgcgtcct cagtcacctt ttgagccaga taattaagag 22740 ccaaatgcaa ttggctcagg ctgccatcgt ccccccgtgc gaaacctgca cgtccgcgtc 22800 aaagaaataa ccggcacctc ttgctgtttt tatcagttga gggcttgacg gatccgcctc 22860 aagtttgcgg cgcagccgca aaatgagaac atctatactc ctgtcgtaaa cctcctcgtc 22920 gcgtactcga ctggcaatga gaagttgctc gcgcgataga acgtcgcggg gtttctctaa 22980 aaacgcgagg agaagattga actcacctgc cgtaagtttc acctcaccgc cagcttcgga 23040 catcaagcga cgttgcctga gattaagtgt ccagtcagta aaacaaaaag accgtcggtc 23100 tttggagcgg acaacgttgg ggcgcacgcg caaggcaacc cgaatgcgtg caagaaactc 23160 tctcgtacta aacggcttag cgataaaatc acttgctcct agctcgagtg caacaacttt 23220 atccgtctcc tcaaggcggt cgccactgat aattatgatt ggaatatcag actttgccgc 23280 cagatttcga acgatctcaa gcccatcttc acgacctaaa tttagatcaa caaccacgac 23340

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   20161011_BB2533PCT_SeqLst.txt atcgaccgtc gcggaagaga gtactctagt gaactgggtg ctgtcggcta ccgcggtcac 23400 tttgaaggcg tggatcgtaa ggtattcgat aataagatgc cgcatagcga catcgtcatc 23460 gataagaaga acgtgtttca acggctcacc tttcaatcta aaatctgaac ccttgttcac 23520 agcgcttgag aaattttcac gtgaaggatg tacaatcatc tccagctaaa tgggcagttc 23580 gtcagaattg cggctgaccg cggatgacga aaatgcgaac caagtatttc aattttatga 23640 caaaagttct caatcgttgt tacaagtgaa acgcttcgag gttacagcta ctattgatta 23700 aggagatcgc ctatggtctc gccccggcgt cgtgcgtccg ccgcgagcca gatctcgcct 23760 acttcataaa cgtcctcata ggcacggaat ggaatgatga catcgatcgc cgtagagagc 23820 atgtcaatca gtgtgcgatc ttccaagcta gcaccttggg cgctactttt gacaagggaa 23880 aacagtttct tgaatccttg gattggattc gcgccgtgta ttgttgaaat cgatcccgga 23940 tgtcccgaga cgacttcact cagataagcc catgctgcat cgtcgcgcat ctcgccaagc 24000 aatatccggt ccggccgcat acgcagactt gcttggagca agtgctcggc gctcacagca 24060 cccagcccag caccgttctt ggagtagagt agtctaacat gattatcgtg tggaatgacg 24120 agttcgagcg tatcttctat ggtgattagc ctttcctggg gggggatggc gctgatcaag 24180 gtcttgctca ttgttgtctt gccgcttccg gtagggccac atagcaacat cgtcagtcgg 24240 ctgacgacgc atgcgtgcag aaacgcttcc aaatccccgt tgtcaaaatg ctgaaggata 24300 gcttcatcat cctgattttg gcgtttcctt cgtgtctgcc actggttcca cctcgaagca 24360 tcataacggg aggagacttc tttaagacca gaaacacgcg agcttggccg tcgaatggtc 24420 aagctgacgg tgcccgaggg aacggtcggc ggcagacaga tttgtagtcg ttcaccacca 24480 ggaagttcag tggcgcagag ggggttacgt ggtccgacat cctgctttct cagcgcgccc 24540 gctaaaatag cgatatcttc aagatcatca taagagacgg gcaaaggcat cttggtaaaa 24600 atgccggctt ggcgcacaaa tgcctctcca ggtcgattga tcgcaatttc ttcagtcttc 24660 gggtcatcga gccattccaa aatcggcttc agaagaaagc gtagttgcgg atccacttcc 24720 atttacaatg tatcctatct ctaagcggaa atttgaattc attaagagcg gcggttcctc 24780 ccccgcgtgg cgccgccagt caggcggagc tggtaaacac caaagaaatc gaggtcccgt 24840 gctacgaaaa tggaaacggt gtcaccctga ttcttcttca gggttggcgg tatgttgatg 24900 gttgccttaa gggctgtctc agttgtctgc tcaccgttat tttgaaagct gttgaagctc 24960 atcccgccac ccgagctgcc ggcgtaggtg ctagctgcct ggaaggcgcc ttgaacaaca 25020 ctcaagagca tagctccgct aaaacgctgc cagaagtggc tgtcgaccga gcccggcaat 25080 cctgagcgac cgagttcgtc cgcgcttggc gatgttaacg agatcatcgc atggtcaggt 25140 gtctcggcgc gatcccacaa cacaaaaacg cgcccatctc cctgttgcaa gccacgctgt 25200 atttcgccaa caacggtggt gccacgatca agaagcacga tattgttcgt tgttccacga 25260 atatcctgag gcaagacaca ctttacatag cctgccaaat ttgtgtcgat tgcggtttgc 25320 aagatgcacg gaattattgt cccttgcgtt accataaaat cggggtgcgg caagagcgtg 25380 gcgctgctgg gctgcagctc ggtgggtttc atacgtatcg acaaatcgtt ctcgccggac 25440 acttcgccat tcggcaagga gttgtcgtca cgcttgcctt cttgtcttcg gcccgtgtcg 25500 ccctgaatgg cgcgtttgct gaccccttga tcgccgctgc tatatgcaaa aatcggtgtt 25560 tcttccggcc gtggctcatg ccgctccggt tcgcccctcg gcggtagagg agcagcaggc 25620 tgaacagcct cttgaaccgc tggaggatcc ggcggcacct caatcggagc tggatgaaat 25680 ggcttggtgt ttgttgcgat caaagttgac ggcgatgcgt tctcattcac cttcttttgg 25740 cgcccaccta gccaaatgag gcttaatgat aacgcgagaa cgacacctcc gacgatcaat 25800 ttctgagacc ccgaaagacg ccggcgatgt ttgtcggaga ccagggatcc agatgcatca 25860 acctcatgtg ccgcttgctg actatcgtta ttcatccctt cgcccccttc aggacgcgtt 25920 tcacatcggg cctcaccgtg cccgtttgcg gcctttggcc aacgggatcg taagcggtgt 25980 tccagataca tagtactgtg tggccatccc tcagacgcca acctcgggaa accgaagaaa 26040 tctcgacatc gctcccttta actgaatagt tggcaacagc ttccttgcca tcaggattga 26100 tggtgtagat ggagggtatg cgtacattgc ccggaaagtg gaataccgtc gtaaatccat 26160 tgtcgaagac ttcgagtggc aacagcgaac gatcgccttg ggcgacgtag tgccaattac 26220 tgtccgccgc accaagggct gtgacaggct gatccaataa attctcagct ttccgttgat 26280 attgtgcttc cgcgtgtagt ctgtccacaa cagccttctg ttgtgcctcc cttcgccgag 26340 ccgccgcatc gtcggcgggg taggcgaatt ggacgctgta atagagatcg ggctgctctt 26400 tatcgaggtg ggacagagtc ttggaactta tactgaaaac ataacggcgc atcccggagt 26460 cgcttgcggt tagcacgatt actggctgag gcgtgaggac ctggcttgcc ttgaaaaata 26520 gataatttcc ccgcggtagg gctgctagat ctttgctatt tgaaacggca accgctgtca 26580 ccgtttcgtt cgtggcgaat gttacgacca aagtagctcc aaccgccgtc gagaggcgca 26640 ccacttgatc gggattgtaa gccaaataac gcatgcgcgg atctagcttg cccgccattg 26700 gagtgtcttc agcctccgca ccagtcgcag cggcaaataa acatgctaaa atgaaaagtg 26760 cttttctgat catggttcgc tgtggcctac gtttgaaacg gtatcttccg atgtctgata 26820 ggaggtgaca accagacctg ccgggttggt tagtctcaat ctgccgggca agctggtcac 26880 cttttcgtag cgaactgtcg cggtccacgt actcaccaca ggcattttgc cgtcaacgac 26940 gagggtcctt ttatagcgaa tttgctgcgt gcttggagtt acatcatttg aagcgatgtg 27000 ctcgacctcc accctgccgc gtttgccaag aatgacttga ggcgaactgg gattgggata 27060 gttgaagaat tgctggtaat cctggcgcac tgttggggca ctgaagttcg ataccaggtc 27120 gtaggcgtac tgagcggtgt cggcatcata actctcgcgc aggcgaacgt actcccacaa 27180 tgaggcgtta acgacggcct cctcttgagt tgcaggcaat cgcgagacag acacctcgct 27240 gtcaacggtg ccgtccggcc gtatccatag atatacgggc acaagcctgc tcaacggcac 27300 cattgtggct atagcgaacg cttgagcaac atttcccaaa atcgcgatag ctgcgacagc 27360 tgcaatgagt ttggagagac gtcgcgccga tttcgctcgc gcggtttgaa aggcttctac 27420

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   20161011_BB2533PCT_SeqLst.txt ttccttatag tgctcggcaa ggctttcgcg cgccactagc atggcatatt caggccccgt 27480 catagcgtcc acccgaattg ccgagctgaa gatctgacgg agtaggctgc catcgcccca 27540 cattcagcgg gaagatcggg cctttgcagc tcgctaatgt gtcgtttgtc tggcagccgc 27600 tcaaagcgac aactaggcac agcaggcaat acttcataga attctccatt gaggcgaatt 27660 tttgcgcgac ctagcctcgc tcaacctgag cgaagcgacg gtacaagctg ctggcagatt 27720 gggttgcgcc gctccagtaa ctgcctccaa tgttgccggc gatcgccggc aaagcgacaa 27780 tgagcgcatc ccctgtcaga aaaaacatat cgagttcgta aagaccaatg atcttggccg 27840 cggtcgtacc ggcgaaggtg attacaccaa gcataagggt gagcgcagtc gcttcggtta 27900 ggatgacgat cgttgccacg aggtttaaga ggagaagcaa gagaccgtag gtgataagtt 27960 gcccgatcca cttagctgcg atgtcccgcg tgcgatcaaa aatatatccg acgaggatca 28020 gaggcccgat cgcgagaagc actttcgtga gaattccaac ggcgtcgtaa actccgaagg 28080 cagaccagag cgtgccgtaa aggacccact gtgccccttg gaaagcaagg atgtcctggt 28140 cgttcatcgg accgatttcg gatgcgattt tctgaaaaac ggcctgggtc acggcgaaca 28200 ttgtatccaa ctgtgccgga acagtctgca gaggcaagcc ggttacacta aactgctgaa 28260 caaagtttgg gaccgtcttt tcgaagatgg aaaccacata gtcttggtag ttagcctgcc 28320 caacaattag agcaacaacg atggtgaccg tgatcacccg agtgataccg ctacgggtat 28380 cgacttcgcc gcgtatgact aaaataccct gaacaataat ccaaagagtg acacaggcga 28440 tcaatggcgc actcaccgcc tcctggatag tctcaagcat cgagtccaag cctgtcgtga 28500 aggctacatc gaagatcgta tgaatggccg taaacggcgc cggaatcgtg aaattcatcg 28560 attggacctg aacttgactg gtttgtcgca taatgttgga taaaatgagc tcgcattcgg 28620 cgaggatgcg ggcggatgaa caaatcgccc agccttaggg gagggcacca aagatgacag 28680 cggtcttttg atgctccttg cgttgagcgg ccgcctcttc cgcctcgtga aggccggcct 28740 gcgcggtagt catcgttaat aggcttgtcg cctgtacatt ttgaatcatt gcgtcatgga 28800 tctgcttgag aagcaaacca ttggtcacgg ttgcctgcat gatattgcga gatcgggaaa 28860 gctgagcaga cgtatcagca ttcgccgtca agcgtttgtc catcgtttcc agattgtcag 28920 ccgcaatgcc agcgctgttt gcggaaccgg tgatctgcga tcgcaacagg tccgcttcag 28980 catcactacc cacgactgca cgatctgtat cgctggtgat cgcacgtgcc gtggtcgaca 29040 ttggcattcg cggcgaaaac atttcattgt ctaggtcctt cgtcgaagga tactgatttt 29100 tctggttgag cgaagtcagt agtccagtaa cgccgtaggc cgacgtcaac atcgtaacca 29160 tcgctatagt ctgagtgaga ttctccgcag tcgcgagcgc agtcgcgagc gtctcagcct 29220 ccgttgccgg gtcgctaaca acaaactgcg cccgcgcggg ctgaatatat agaaagctgc 29280 aggtcaaaac tgttgcaata agttgcgtcg tcttcatcgt ttcctacctt atcaatcttc 29340 tgcctcgtgg tgacgggcca tgaattcgct gagccagcca gatgagttgc cttcttgtgc 29400 ctcgcgtagt cgagttgcaa agcgcaccgt gttggcacgc cccgaaagca cggcgacata 29460 ttcacgcata tcccgcagat caaattcgca gatgacgctt ccactttctc gtttaagaag 29520 aaacttacgg ctgccgaccg tcatgtcttc acggatcgcc tgaaattcct tttcggtaca 29580 tttcagtcca tcgacataag ccgatcgatc tgcggttggt gatggataga aaatcttcgt 29640 catacattgc gcaaccaagc tggctcctag cggcgattcc agaacatgct ctggttgctg 29700 cgttgccagt attagcatcc cgttgttttt tcgaacggtc aggaggaatt tgtcgacgac 29760 agtcgaaaat ttagggttta acaaataggc gcgaaactca tcgcagctca tcacaaaacg 29820 gcggccgtcg atcatggctc caatccgatg caggagatat gctgcagcgg gagcgcatac 29880 ttcctcgtat tcgagaagat gcgtcatgtc gaagccggta atcgacggat ctaactttac 29940 ttcgtcaact tcgccgtcaa atgcccagcc aagcgcatgg ccccggcacc agcgttggag 30000 ccgcgctcct gcgccttcgg cgggcccatg caacaaaaat tcacgtaacc ccgcgattga 30060 acgcatttgt ggatcaaacg agagctgacg atggatacca cggaccagac ggcggttctc 30120 ttccggagaa atcccacccc gaccatcact ctcgatgaga gccacgatcc attcgcgcag 30180 aaaatcgtgt gaggctgctg tgttttctag gccacgcaac ggcgccaacc cgctgggtgt 30240 gcctctgtga agtgccaaat atgttcctcc tgtggcgcga accagcaatt cgccaccccg 30300 gtccttgtca aagaacacga ccgtacctgc acggtcgacc atgctctgtt cgagcatggc 30360 tagaacaaac atcatgagcg tcgtcttacc cctcccgata ggcccgaata ttgccgtcat 30420 gccaacatcg tgctcatgcg ggatatagtc gaaaggcgtt ccgccattgg tacgaaatcg 30480 ggcaatcgcg ttgccccagt ggcctgagct ggcgccctct ggaaagtttt cgaaagagac 30540 aaaccctgcg aaattgcgtg aagtgattgc gccagggcgt gtgcgccact taaaattccc 30600 cggcaattgg gaccaatagg ccgcttccat accaatacct tcttggacaa ccacggcacc 30660 tgcatccgcc attcgtgtcc gagcccgcgc gcccctgtcc ccaagactat tgagatcgtc 30720 tgcatagacg caaaggctca aatgatgtga gcccataacg aattcgttgc tcgcaagtgc 30780 gtcctcagcc tcggataatt tgccgatttg agtcacggct ttatcgccgg aactcagcat 30840 ctggctcgat ttgaggctaa gtttcgcgtg cgcttgcggg cgagtcagga acgaaaaact 30900 ctgcgtgaga acaagtggaa aatcgaggga tagcagcgcg ttgagcatgc ccggccgtgt 30960 ttttgcaggg tattcgcgaa acgaatagat ggatccaacg taactgtctt ttggcgttct 31020 gatctcgagt cctcgcttgc cgcaaatgac tctgtcggta taaatcgaag cgccgagtga 31080 gccgctgacg accggaaccg gtgtgaaccg accagtcatg atcaaccgta gcgcttcgcc 31140 aatttcggtg aagagcacac cctgcttctc gcggatgcca agacgatgca ggccatacgc 31200 tttaagagag ccagcgacaa catgccaaag atcttccatg ttcctgatct ggcccgtgag 31260 atcgttttcc ctttttccgc ttagcttggt gaacctcctc tttaccttcc ctaaagccgc 31320 ctgtgggtag acaatcaacg taaggaagtg ttcattgcgg aggagttggc cggagagcac 31380 gcgctgttca aaagcttcgt tcaggctagc ggcgaaaaca ctacggaagt gtcgcggcgc 31440 cgatgatggc acgtcggcat gacgtacgag gtgagcatat attgacacat gatcatcagc 31500

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   20161011_BB2533PCT_SeqLst.txt gatattgcgc aacagcgtgt tgaacgcacg acaacgcgca ttgcgcattt cagtttcctc 31560 aagctcgaat gcaacgccat caattctcgc aatggtcatg atcgatccgt cttcaagaag 31620 gacgatatgg tcgctgaggt ggccaatata agggagatag atctcaccgg atctttcggt 31680 cgttccactc gcgccgagca tcacaccatt cctctccctc gtgggggaac cctaattgga 31740 tttgggctaa cagtagcgcc cccccaaact gcactatcaa tgcttcttcc cgcggtccgc 31800 aaaaatagca ggacgacgct cgccgcattg tagtctcgct ccacgatgag ccgggctgca 31860 aaccataacg gcacgagaac gacttcgtag agcgggttct gaacgataac gatgacaaag 31920 ccggcgaaca tcatgaataa ccctgccaat gtcagtggca ccccaagaaa caatgcgggc 31980 cgtgtggctg cgaggtaaag ggtcgattct tccaaacgat cagccatcaa ctaccgccag 32040 tgagcgtttg gccgaggaag ctcgccccaa acatgataac aatgccgccg acgacgccgg 32100 caaccagccc aagcgaagcc cgcccgaaca tccaggagat cccgatagcg acaatgccga 32160 gaacagcgag tgactggccg aacggaccaa ggataaacgt gcatatattg ttaaccattg 32220 tggcggggtc agtgccgcca cccgcagatt gcgctgcggc gggtccggat gaggaaatgc 32280 tccatgcaat tgcaccgcac aagcttgggg cgcagctcga tatcacgcgc atcatcgcat 32340 tcgagagcga gaggcgattt agatgtaaac ggtatctctc aaagcatcgc atcaatgcgc 32400 acctccttag tataagtcga ataagacttg attgtcgtct gcggatttgc cgttgtcctg 32460 gtgtggcggt ggcggagcga ttaaaccgcc agcgccatcc tcctgcgagc ggcgctgata 32520 tgacccccaa acatcccacg tctcttcgga ttttagcgcc tcgtgatcgt cttttggagg 32580 ctcgattaac gcgggcacca gcgattgagc agctgtttca acttttcgca cgtagccgtt 32640 tgcaaaaccg ccgatgaaat taccggtgtt gtaagcggag atcgcccgac gaagcgcaaa 32700 ttgcttctcg tcaatcgttt cgccgcctgc ataacgactt ttcagcatgt ttgcagcggc 32760 agataatgat gtgcacgcct ggagcgcacc gtcaggtgtc agaccgagca tagaaaaatt 32820 tcgagagttt atttgcatga ggccaacatc cagcgaatgc cgtgcatcga gacggtgcct 32880 gacgacttgg gttgcttggc tgtgatcttg ccagtgaagc gtttcgccgg tcgtgttgtc 32940 atgaatcgct aaaggatcaa agcgactctc caccttagct atcgccgcaa gcgtagatgt 33000 cgcaactgat ggggcacact tgcgagcaac atggtcaaac tcagcagatg agagtggcgt 33060 ggcaaggctc gacgaacaga aggagaccat caaggcaaga gaaagcgacc ccgatctctt 33120 aagcatacct tatctcctta gctcgcaact aacaccgcct ctcccgttgg aagaagtgcg 33180 ttgttttatg ttgaagatta tcgggagggt cggttactcg aaaattttca attgcttctt 33240 tatgatttca attgaagcga gaaacctcgc ccggcgtctt ggaacgcaac atggaccgag 33300 aaccgcgcat ccatgactaa gcaaccggat cgacctattc aggccgcagt tggtcaggtc 33360 aggctcagaa cgaaaatgct cggcgaggtt acgctgtctg taaacccatt cgatgaacgg 33420 gaagcttcct tccgattgct cttggcagga atattggccc atgcctgctt gcgctttgca 33480 aatgctctta tcgcgttggt atcatatgcc ttgtccgcca gcagaaacgc actctaagcg 33540 attatttgta aaaatgtttc ggtcatgcgg cggtcatggg cttgacccgc tgtcagcgca 33600 agacggatcg gtcaaccgtc ggcatcgaca acagcgtgaa tcttggtggt caaaccgcca 33660 cgggaacgtc ccatacagcc atcgtcttga tcccgctgtt tcccgtcgcc gcatgttggt 33720 ggacgcggac acaggaactg tcaatcatga cgacattcta tcgaaagcct tggaaatcac 33780 actcagaata tgatcccaga cgtctgcctc acgccatcgt acaaagcgat tgtagcaggt 33840 tgtacaggaa ccgtatcgat caggaacgtc tgcccagggc gggcccgtcc ggaagcgcca 33900 caagatgaca ttgatcaccc gcgtcaacgc gcggcacgcg acgcggctta tttgggaaca 33960 aaggactgaa caacagtcca ttcgaaatcg gtgacatcaa agcggggacg ggttatcagt 34020 ggcctccaag tcaagcctca atgaatcaaa atcagaccga tttgcaaacc tgatttatga 34080 gtgtgcggcc taaatgatga aatcgtcctt ctagatcgcc tccgtggtgt agcaacacct 34140 cgcagtatcg ccgtgctgac cttggccagg gaattgactg gcaagggtgc tttcacatga 34200 ccgctctttt ggccgcgata gatgatttcg ttgctgcttt gggcacgtag aaggagagaa 34260 gtcatatcgg agaaattcct cctggcgcga gagcctgctc tatcgcgacg gcatcccact 34320 gtcgggaaca gaccggatca ttcacgaggc gaaagtcgtc aacacatgcg ttataggcat 34380 cttcccttga aggatgatct tgttgctgcc aatctggagg tgcggcagcc gcaggcagat 34440 gcgatctcag cgcaacttgc ggcaaaacat ctcactcacc tgaaaaccac tagcgagtct 34500 cgcgatcaga cgaaggcctt ttacttaacg acacaatatc cgatgtctgc atcacaggcg 34560 tcgctatccc agtcaatact aaagcggtgc aggaactaaa gattactgat gacttaggcg 34620 tgccacgagg cctgagacga cgcgcgtaga cagttttttg aaatcattat caaagtgatg 34680 gcctccgctg aagcctatca cctctgcgcc ggtctgtcgg agagatgggc aagcattatt 34740 acggtcttcg cgcccgtaca tgcattggac gattgcaggg tcaatggatc tgagatcatc 34800 cagaggattg ccgcccttac cttccgtttc gagttggagc cagcccctaa atgagacgac 34860 atagtcgact tgatgtgaca atgccaagag agagatttgc ttaacccgat ttttttgctc 34920 aagcgtaagc ctattgaagc ttgccggcat gacgtccgcg ccgaaagaat atcctacaag 34980 taaaacattc tgcacaccga aatgcttggt gtagacatcg attatgtgac caagatcctt 35040 agcagtttcg cttggggacc gctccgacca gaaataccga agtgaactga cgccaatgac 35100 aggaatccct tccgtctgca gataggtacc atcgatagat ctgctgcctc gcgcgtttcg 35160 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 35220 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc 35280 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc 35340 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg 35400 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg 35460 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 35520 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 35580

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   20161011_BB2533PCT_SeqLst.txt gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 35640 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 35700 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 35760 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 35820 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 35880 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 35940 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 36000 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 36060 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 36120 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 36180 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 36240 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 36300 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 36360 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 36420 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 36480 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 36540 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 36600 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 36660 tttgcgcaac gttgttgcca ttgctgcagg gggggggggg ggggggttcc attgttcatt 36720 ccacggacaa aaacagagaa aggaaacgac agaggccaaa aagctcgctt tcagcacctg 36780 tcgtttcctt tcttttcaga gggtatttta aataaaaaca ttaagttatg acgaagaaga 36840 acggaaacgc cttaaaccgg aaaattttca taaatagcga aaacccgcga ggtcgccgcc 36900 ccgtaacctg tcggatcacc ggaaaggacc cgtaaagtga taatgattat catctacata 36960 tcacaacgtg cgtggaggcc atcaaaccac gtcaaataat caattatgac gcaggtatcg 37020 tattaattga tctgcatcaa cttaacgtaa aaacaacttc agacaataca aatcagcgac 37080 actgaatacg gggcaacctc atgtcccccc cccccccccc cctgcaggca tcgtggtgtc 37140 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 37200 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 37260 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 37320 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 37380 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc 37440 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 37500 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 37560 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 37620 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 37680 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 37740 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 37800 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 37860 ctttcgtctt caagaattgg tcgacgatct tgctgcgttc ggatattttc gtggagttcc 37920 cgccacagac ccggattgaa ggcgagatcc agcaactcgc gccagatcat cctgtgacgg 37980 aactttggcg cgtgatgact ggccaggacg tcggccgaaa gagcgacaag cagatcacgc 38040 ttttcgacag cgtcggattt gcgatcgagg atttttcggc gctgcgctac gtccgcgacc 38100 gcgttgaggg atcaagccac agcagcccac tcgaccttct agccgaccca gacgagccaa 38160 gggatctttt tggaatgctg ctccgtcgtc aggctttccg acgtttgggt ggttgaacag 38220 aagtcattat cgtacggaat gccaagcact cccgagggga accctgtggt tggcatgcac 38280 atacaaatgg acgaacggat aaaccttttc acgccctttt aaatatccgt tattctaata 38340 aacgctcttt tctcttaggt ttacccgcca atatatcctg tcaaacactg atagtttaaa 38400 ctgaaggcgg gaaacgacaa tctgatcatg agcggagaat taagggagtc acgttatgac 38460 ccccgccgat gacgcgggac aagccgtttt acgtttggaa ctgacagaac cgcaacgttg 38520 aaggagccac tcagcccaag ctggtacgat tgtaatacga ctcactatag ggcgaattga 38580 gcgctgttta aacgctcttc aactggaaga gcggttacca gaggccagaa tggccatctc 38640 ggaccgatat cgctatcaac tttgtataga aaagttgggc cgaattcgag ctcggtacgg 38700 ccagaatggc ccggaccggg ttaccgaatt cgagctcggt accctgggat ccgatatcga 38760 tgggccctgg ccgaagcttg catgcctgca gtgcagcgtg acccggtcgt gcccctctct 38820 agagataatg agcattgcat gtctaagtta taaaaaatta ccacatattt tttttgtcac 38880 acttgtttga agtgcagttt atctatcttt atacatatat ttaaacttta ctctacgaat 38940 aatataatct atagtactac aataatatca gtgttttaga gaatcatata aatgaacagt 39000 tagacatggt ctaaaggaca attgagtatt ttgacaacag gactctacag ttttatcttt 39060 ttagtgtgca tgtgttctcc tttttttttg caaatagctt cacctatata atacttcatc 39120 cattttatta gtacatccat ttagggttta gggttaatgg tttttataga ctaatttttt 39180 tagtacatct attttattct attttagcct ctaaattaag aaaactaaaa ctctatttta 39240 gtttttttat ttaataattt agatataaaa tagaataaaa taaagtgact aaaaattaaa 39300 caaataccct ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt cgagtagata 39360 atgccagcct gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc 39420 gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct gtcgctgcct ctggacccct 39480 ctcgagagtt ccgctccacc gttggacttg ctccgctgtc ggcatccaga aattgcgtgg 39540 cggagcggca gacgtgagcc ggcacggcag gcggcctcct cctcctctca cggcaccggc 39600 agctacgggg gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata 39660

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   20161011_BB2533PCT_SeqLst.txt aatagacacc ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca 39720 cacaaccaga tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg 39780 tcctcccccc cccccctctc taccttctct agatcggcgt tccggtccat gcatggttag 39840 ggcccggtag ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt 39900 gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt 39960 gccagtgttt ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga 40020 tttcatgatt ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa 40080 tatatgccgt gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg 40140 atgatgtggt ctggttgggc ggtcgttcta gatcggagta gaattctgtt tcaaactacc 40200 tggtggattt attaattttg gatctgtatg tgtgtgccat acatattcat agttacgaat 40260 tgaagatgat ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac 40320 tgatgcatat acagagatgc tttttgttcg cttggttgtg atgatgtggt gtggttgggc 40380 ggtcgttcat tcgttctaga tcggagtaga atactgtttc aaactacctg gtgtatttat 40440 taattttgga actgtatgtg tgtgtcatac atcttcatag ttacgagttt aagatggatg 40500 gaaatatcga tctaggatag gtatacatgt tgatgtgggt tttactgatg catatacatg 40560 atggcatatg cagcatctat tcatatgctc taaccttgag tacctatcta ttataataaa 40620 caagtatgtt ttataattat tttgatcttg atatacttgg atgatggcat atgcagcagc 40680 tatatgtgga tttttttagc cctgccttca tacgctattt atttgcttgg tactgtttct 40740 tttgtcgatg ctcaccctgt tgtttggtgt tacttctgca ggtcgactct agaggatcca 40800 tggcaccgaa gaagaagcgc aaggtgatgg acaagaagta cagcatcggc ctcgacatcg 40860 gcaccaactc ggtgggctgg gccgtcatca cggacgaata taaggtcccg tcgaagaagt 40920 tcaaggtcct cggcaataca gaccgccaca gcatcaagaa aaacttgatc ggcgccctcc 40980 tgttcgatag cggcgagacc gcggaggcga ccaggctcaa gaggaccgcc aggagacggt 41040 acactaggcg caagaacagg atctgctacc tgcaggagat cttcagcaac gagatggcga 41100 aggtggacga ctccttcttc caccgcctgg aggaatcatt cctggtggag gaggacaaga 41160 agcatgagcg gcacccaatc ttcggcaaca tcgtcgacga ggtaagtttc tgcttctacc 41220 tttgatatat atataataat tatcattaat tagtagtaat ataatatttc aaatattttt 41280 ttcaaaataa aagaatgtag tatatagcaa ttgcttttct gtagtttata agtgtgtata 41340 ttttaattta taacttttct aatatatgac caaaacatgg tgatgtgcag gtggcctacc 41400 acgagaagta cccgacaatc taccacctcc ggaagaaact ggtggacagc acagacaagg 41460 cggacctccg gctcatctac cttgccctcg cgcatatgat caagttccgc ggccacttcc 41520 tcatcgaggg cgacctgaac ccggacaact ccgacgtgga caagctgttc atccagctcg 41580 tgcagacgta caatcaactg ttcgaggaga accccataaa cgctagcggc gtggacgcca 41640 aggccatcct ctcggccagg ctctcgaaat caagaaggct ggagaacctt atcgcgcagt 41700 tgccaggcga aaagaagaac ggcctcttcg gcaaccttat tgcgctcagc ctcggcctga 41760 cgccgaactt caaatcaaac ttcgacctcg cggaggacgc caagctccag ctctcaaagg 41820 acacctacga cgacgacctc gacaacctcc tggcccagat aggagaccag tacgcggacc 41880 tcttcctcgc cgccaagaac ctctccgacg ctatcctgct cagcgacatc cttcgggtca 41940 acaccgaaat taccaaggca ccgctgtccg ccagcatgat taaacgctac gacgagcacc 42000 atcaggacct cacgctgctc aaggcactcg tccgccagca gctccccgag aagtacaagg 42060 agatcttctt cgaccaatca aaaaacggct acgcgggata tatcgacggc ggtgccagcc 42120 aggaagagtt ctacaagttc atcaaaccaa tcctggagaa gatggacggc accgaggagt 42180 tgctggtcaa gctcaacagg gaggacctcc tcaggaagca gaggaccttc gacaacggct 42240 ccatcccgca tcagatccac ctgggcgaac tgcatgccat cctgcggcgc caggaggact 42300 tctacccgtt cctgaaggat aaccgggaga agatcgagaa gatcttgacg ttccgcatcc 42360 catactacgt gggcccgctg gctcgcggca actcccggtt cgcctggatg acccggaagt 42420 cggaggagac catcacaccc tggaactttg aggaggtggt cgataagggc gctagcgctc 42480 agagcttcat cgagcgcatg accaacttcg ataaaaacct gcccaatgaa aaagtcctcc 42540 ccaagcactc gctgctctac gagtacttca ccgtgtacaa cgagctcacc aaggtcaaat 42600 acgtcaccga gggcatgcgg aagccggcgt tcctgagcgg cgagcagaag aaggcgatag 42660 tggacctcct cttcaagacc aacaggaagg tgaccgtgaa gcaattaaaa gaggactact 42720 tcaagaaaat agagtgcttc gactccgtgg agatctcggg cgtggaggat cggttcaacg 42780 cctcactcgg cacgtatcac gacctcctca agatcattaa agacaaggac ttcctcgaca 42840 acgaggagaa cgaggacatc ctcgaggaca tcgtcctcac cctgaccctg ttcgaggacc 42900 gcgaaatgat cgaggagagg ctgaagacct acgcgcacct gttcgacgac aaggtcatga 42960 aacagctcaa gaggcgccgc tacactggtt ggggaaggct gtcccgcaag ctcattaatg 43020 gcatcaggga caagcagagc ggcaagacca tcctggactt cctcaagtcc gacgggttcg 43080 ccaaccgcaa cttcatgcag ctcattcacg acgactcgct cacgttcaag gaagacatcc 43140 agaaggcaca ggtgagcggg cagggtgact ccctccacga acacatcgcc aacctggccg 43200 gctcgccggc cattaaaaag ggcatcctgc agacggtcaa ggtcgtcgac gagctcgtga 43260 aggtgatggg ccggcacaag cccgaaaata tcgtcataga gatggccagg gagaaccaga 43320 ccacccaaaa agggcagaag aactcgcgcg agcggatgaa acggatcgag gagggcatta 43380 aagagctcgg gtcccagatc ctgaaggagc accccgtgga aaatacccag ctccagaatg 43440 aaaagctcta cctctactac ctgcagaacg gccgcgacat gtacgtggac caggagctgg 43500 acattaatcg gctatcggac tacgacgtcg accacatcgt gccgcagtcg ttcctcaagg 43560 acgatagcat cgacaacaag gtgctcaccc ggtcggataa aaatcggggc aagagcgaca 43620 acgtgcccag cgaggaggtc gtgaagaaga tgaaaaacta ctggcgccag ctcctcaacg 43680 cgaaactgat cacccagcgc aagttcgaca acctgacgaa ggcggaacgc ggtggcttga 43740

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   20161011_BB2533PCT_SeqLst.txt gcgaactcga taaggcgggc ttcataaaaa ggcagctggt cgagacgcgc cagatcacga 43800 agcatgtcgc ccagatcctg gacagccgca tgaatactaa gtacgatgaa aacgacaagc 43860 tgatccggga ggtgaaggtg atcacgctga agtccaagct cgtgtcggac ttccgcaagg 43920 acttccagtt ctacaaggtc cgcgagatca acaactacca ccacgcccac gacgcctacc 43980 tgaatgcggt ggtcgggacc gccctgatca agaagtaccc gaagctggag tcggagttcg 44040 tgtacggcga ctacaaggtc tacgacgtgc gcaaaatgat cgccaagtcc gagcaggaga 44100 tcggcaaggc cacggcaaaa tacttcttct actcgaacat catgaacttc ttcaagaccg 44160 agatcaccct cgcgaacggc gagatccgca agcgcccgct catcgaaacc aacggcgaga 44220 cgggcgagat cgtctgggat aagggccggg atttcgcgac ggtccgcaag gtgctctcca 44280 tgccgcaagt caatatcgtg aaaaagacgg aggtccagac gggcgggttc agcaaggagt 44340 ccatcctccc gaagcgcaac tccgacaagc tcatcgcgag gaagaaggat tgggacccga 44400 aaaaatatgg cggcttcgac agcccgaccg tcgcatacag cgtcctcgtc gtggcgaagg 44460 tggagaaggg caagtcaaag aagctcaagt ccgtgaagga gctgctcggg atcacgatta 44520 tggagcggtc ctccttcgag aagaacccga tcgacttcct agaggccaag ggatataagg 44580 aggtcaagaa ggacctgatt attaaactgc cgaagtactc gctcttcgag ctggaaaacg 44640 gccgcaagag gatgctcgcc tccgcaggcg agttgcagaa gggcaacgag ctcgccctcc 44700 cgagcaaata cgtcaatttc ctgtacctcg ctagccacta tgaaaagctc aagggcagcc 44760 cggaggacaa cgagcagaag cagctcttcg tggagcagca caagcattac ctggacgaga 44820 tcatcgagca gatcagcgag ttctcgaagc gggtgatcct cgccgacgcg aacctggaca 44880 aggtgctgtc ggcatataac aagcaccgcg acaaaccaat acgcgagcag gccgaaaata 44940 tcatccacct cttcaccctc accaacctcg gcgctccggc agccttcaag tacttcgaca 45000 ccacgattga ccggaagcgg tacacgagca cgaaggaggt gctcgatgcg acgctgatcc 45060 accagagcat cacagggctc tatgaaacac gcatcgacct gagccagctg ggcggagaca 45120 agagaccacg ggaccgccac gatggcgagc tgggaggccg caagcgggca aggtaggtac 45180 cgttaaccta gacttgtcca tcttctggat tggccaactt aattaatgta tgaaataaaa 45240 ggatgcacac atagtgacat gctaatcact ataatgtggg catcaaagtt gtgtgttatg 45300 tgtaattact agttatctga ataaaagaga aagagatcat ccatatttct tatcctaaat 45360 gaatgtcacg tgtctttata attctttgat gaaccagatg catttcatta accaaatcca 45420 tatacatata aatattaatc atatataatt aatatcaatt gggttagcaa aacaaatcta 45480 gtctaggtgt gttttgcgaa tgcggccgcc accgcggtgg agctcgaatt cgagctcggt 45540 accctgggat ccagcttcgc ttagttttta gtttttggca gaaaaaatga tcaatgtttc 45600 acaaaccaaa tatttttata acttttgatg aaagaagatc accacggtca tatctagggg 45660 tggtaacaaa ttgcgatcta aatgtttctt cataaaaaat aaggcttctt aataaatttt 45720 agttcaaaat aaatacgaat aaagtctgat tctaatctga ttcgatcctt aaattttata 45780 atgcaaaatt tagagctcat taccacctct agtcatatgt ctagtctgag gtatatccaa 45840 aaagcccttt ctctaaattc cacacccaac tcagatgttt gcaaataaat actccgactc 45900 caaaatgtag gtgaagtgca actttctcca ttttatatca acatttgtta ttttttgttt 45960 aacatttcac actcaaaact aattaataaa atacgtggtt gttgaacgtg cgcacatgtc 46020 tcccttacat tatgtttttt tatttatgta ttattgttgt tttcctccga acaacttgtc 46080 aacatatcat cattggtctt taatatttat gaatatggaa gcctagttat ttacacttgg 46140 ctacacacta gttgtagttt tgccacttgt ctaacatgca actctagtag ttttgccact 46200 tgcctggcat gcaactctag tattgacact tgtatagcat ataatgccaa tacgacacct 46260 gccttacatg aaacattatt tttgacactt gtataccatg caacattacc attgacattt 46320 gtccatacac attatatcaa atatattgag cgcatgtcac aaactcgata caaagctgga 46380 tgaccctccc tcaccacatc tataaaaacc cgagcgctac tgtaaatcac tcacaacaca 46440 acacatatct tttagtaacc tttcaatagg cgtcccccaa gaactagtaa ccatggccct 46500 gtccaacaag ttcatcggcg acgacatgaa gatgacctac cacatggacg gctgcgtgaa 46560 cggccactac ttcaccgtga agggcgaggg cagcggcaag ccctacgagg gcacccagac 46620 ctccaccttc aaggtgacca tggccaacgg cggccccctg gccttctcct tcgacatcct 46680 gtccaccgtg ttcatgtacg gcaaccgctg cttcaccgcc taccccacca gcatgcccga 46740 ctacttcaag caggccttcc ccgacggcat gtcctacgag agaaccttca cctacgagga 46800 cggcggcgtg gccaccgcca gctgggagat cagcctgaag ggcaactgct tcgagcacaa 46860 gtccaccttc cacggcgtga acttccccgc cgacggcccc gtgatggcca agaagaccac 46920 cggctgggac ccctccttcg agaagatgac cgtgtgcgac ggcatcttga agggcgacgt 46980 gaccgccttc ctgatgctgc agggcggcgg caactacaga tgccagttcc acacctccta 47040 caagaccaag aagcccgtga ccatgccccc caaccacgtg gtggagcacc gcatcgccag 47100 aaccgacctg gacaagggcg gcaacagcgt gcagctgacc gagcacgccg tggcccacat 47160 cacctccgtg gtgcccttct gaagcggccc atggatattc gaacgcgtag gtaccacatg 47220 gttaacctag acttgtccat cttctggatt ggccaactta attaatgtat gaaataaaag 47280 gatgcacaca tagtgacatg ctaatcacta taatgtgggc atcaaagttg tgtgttatgt 47340 gtaattacta gttatctgaa taaaagagaa agagatcatc catatttctt atcctaaatg 47400 aatgtcacgt gtctttataa ttctttgatg aaccagatgc atttcattaa ccaaatccat 47460 atacatataa atattaatca tatataatta atatcaattg ggttagcaaa acaaatctag 47520 tctaggtgtg ttttgcgaat tcccatggac ctcgaggggg ggcccgggca cccagctttc 47580 ttgtacaaag tggccgttaa cggatcggcc agaatggccc ggaccgggtt accgaattcg 47640 agctcggtac cctgggatcg gccgctctag aactagtgga tcccccgggc tgcaggaatt 47700 cccatggagt caaagattca aatagaggac ctaacagaac tcgccgtaaa gactggcgaa 47760 cagttcatac agagtctctt acgactcaat gacaagaaga aaatcttcgt caacatggtg 47820

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   20161011_BB2533PCT_SeqLst.txt gagcacgaca cgcttgtcta ctccaaaaat atcaaagata cagtctcaga agaccaaagg 47880 gcaattgaga cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca 47940 gctatctgtc actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat 48000 cattgcgata aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat 48060 ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag 48120 caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactaagct 48180 tcggccgggg cccatcgatc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 48240 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agctcagatc 48300 agcttggggc tggtatcgat aaatgtttcc acatagattt tgcatatcat aatgatgttt 48360 gtcgttccgt atctatgttt catacaaaat ttttacgcat atcgcaacac atgggcacat 48420 acctagtgac tgtataactc tgcatgtatg agtgtatgac tatatgatgt agtaactaat 48480 aagaagggta gacatttgag tgattctttt attcctggac ttgtaagact tgacatttct 48540 gccttgagtg cgatacatca tatggacagg ggttatgcat acactgcttg tttgttgttt 48600 atgttctaag agcatctcca acaacgtgac atatgaaaat gccctacaat ttaaaaatgg 48660 ttatatttta taaaatttag ggcataaata aaacatcccg ctccaacatt aaagccttaa 48720 atctattata gggaagccca ctatgatata gtatatttga ggcactttag agggtgccct 48780 ataatttttt gaccattttt ttatgaaatg agacactatt ggagtatttt ttttccgtag 48840 agcaccatat ttcaatttga gacaccaatt taaggcattg ttggagatgt tctaaatgtt 48900 ggtttatttt gtctgtatcg ttgtggtttt gatagtggtg cctttgcaat gtacatctta 48960 cattgacaat aataataggt aaaactctac aaatttttta tctaatggac tcttgtatga 49020 aacattgtac ttgcacacat ctgatgtaaa cactgcatac ttttaacagt gacaagattc 49080 tgtttcattt tagggctagt ttgggaacca aattttatta gggtttttat tttctaagaa 49140 aaagtaattt attttacctt gagaaaatat aaattacttg agaaaataga gttccaaact 49200 agctcttatc tttgtcgaat cctcctctat tcaaatgtga catttctggc acgtgacaac 49260 tggtgatgtt gtagactgtg ttaagtaata cgtgtcatta ttactaaatg ccattttagt 49320 aaatgttgag tatgtactct actacagtaa gtattattgg tgtatttaca ctagacagtt 49380 ggcggcctgg cgggtaaagt tatcctgtag aaagttgggc caggccaaaa ccaaccgcca 49440 aaggaaaggc cttccggccc gcccaccttt gcgcgccgaa ggtcagttcc ttcagtctcc 49500 tcccgcttca gactctgacc acgtcgacaa tccgggccga aacacatctg caccgtccac 49560 ttgcgacaga ttgaacacac cacttctatc cacgtcagcg atccgtggca ctagcccttc 49620 caccaatcag cccaagttgc ccctttcctt taaattcgcc gcacccattg ctcttctcac 49680 ggccatagaa atcgaccgag cgaatccctc gcatcgcatt cgcagccttt gctgcatcac 49740 accaccgcga aaccccagca gccgcatctg caggtcgact ctagaggatc catggcctcc 49800 tccgaggacg tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac 49860 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc 49920 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc 49980 cagttccagt acggctccaa ggtgtacgtg aagcaccccg ccgacatccc cgactacaag 50040 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc 50100 gtggtgacag tgacccagga ctcctccctg caggacggct ccttcatcta caaggtgaag 50160 ttcatcggcg tgaacttccc ctccgacggc cccgtaatgc agaagaagac tatgggctgg 50220 gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccacaag 50280 gccctgaagc tgaaggacgg cggccacgct agcccatcca cccactcact cactcatatc 50340 tgtgctgtac gtacgagaat ttctcgacca accgtcgtga gacctgccca ccggagatcg 50400 gacgcaagag ggtttaggca agaatgtcgt gcgacagggt gagcgctgac tagtatacgt 50460 gagagacctt gagatatacc tcacacgtac gcgtacttta catgacgtag gacattacga 50520 ctcaaacaga ttcacgtcag atttcggagt ttctcacgcg tgagagcctt ggagggcggt 50580 atgtatgtca tactatatgt tgggatggag ggagtgagtg agtgatatgt ggctagcaag 50640 ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttccagta cggctccaag 50700 gtgtacgtga agcaccccgc cgacatcccc gactacaaga agctgtcctt ccccgagggc 50760 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgacagt gacccaggac 50820 tcctccctgc aggacggctc cttcatctac aaggtgaagt tcatcggcgt gaacttcccc 50880 tccgacggcc ccgtaatgca gaagaagact atgggctggg aggcctccac cgagcgcctg 50940 tacccccgcg acggcgtgct gaagggcgag atccacaagg ccctgaagct gaaggacggc 51000 ggccactacc tggtggagtt caagtccatc tacatggcca agaagcccgt gcagctgccc 51060 ggctactact acgtggactc caagctggac atcacctccc acaacgagga ctacaccatc 51120 gtggagcagt acgagcgcgc cgagggccgc caccacctgt tcctgtagtc aggatctgag 51180 tcgaaaccta gacttgtcca tcttctggat tggccaactt aattaatgta tgaaataaaa 51240 ggatgcacac atagtgacat gctaatcact ataatgtggg catcaaagtt gtgtgttatg 51300 tgtaattact agttatctga ataaaagaga aagagatcat ccatatttct tatcctaaat 51360 gaatgtcacg tgtctttata attctttgat gaaccagatg catttcatta accaaatcca 51420 tatacatata aatattaatc atatataatt aatatcaatt gggttagcaa aacaaatcta 51480 gtctaggtgt gttttgcgaa tgcggccgcc accgcggtgg agctcgaatt ccggtccggg 51540 tcacccggtc cgggcctaga aggccagctt gcggccgccc cgggcaactt tattatacaa 51600 agttgataga tatcggtccg agcggcctag aaggcctttg gtcacctttg tccaccaaga 51660 tggaactgcg gccgctcatt aattaagtca ggcgcgcctc tagttgaaga cacgttcatg 51720 tcttcatcgt aagaagacac tcagtagtct tcggccagaa tggcctaact caaggccatc 51780 gtggcctctt gctcttcagg atgaagagct atgtttaaac gtgcaagcgc tactagacaa 51840 ttcagtacat taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta 51900

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   20161011_BB2533PCT_SeqLst.txt caccacaata tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa 51960 aatcaccact cgatacaggc agcccatcag tccgggacgg cgtcagcggg agagccgttg 52020 taaggcggca gactttgctc atgttaccga tgctattcgg aagaacggca actaagctgc 52080 cgggtttgaa acacggatga tctcgcggag ggtagcatgt tgattgtaac gatgacagag 52140 cgttgctgcc tgtgatcaaa tatcatctcc ctcgcagaga tccgaattat cagccttctt 52200 attcatttct cgcttaaccg tgacaggctg tcgatcttga gaactatgcc gacataatag 52260 gaaatcgctg gataaagccg ctgaggaagc tgagtggcgc tatttcttta gaagtgaacg 52320 ttgacgatcg tcgaccgtac cccgatgaat taattcggac gtacgttctg aacacagctg 52380 gatacttact tgggcgattg tcatacatga catcaacaat gtacccgttt gtgtaaccgt 52440 ctcttggagg ttcgtatgac actagtggtt cccctcagct tgcgactaga tgttgaggcc 52500 taacatttta ttagagagca ggctagttgc ttagatacat gatcttcagg ccgttatctg 52560 tcagggcaag cgaaaattgg ccatttatga cgaccaatgc cccgcagaag ctcccatctt 52620 tgccgccata gacgccgcgc cccccttttg gggtgtagaa catccttttg ccagatgtgg 52680 aaaagaagtt cgttgtccca ttgttggcaa tgacgtagta gccggcgaaa gtgcgagacc 52740 catttgcgct atatataagc ctacgatttc cgttgcgact attgtcgtaa ttggatgaac 52800 tattatcgta gttgctctca gagttgtcgt aatttgatgg actattgtcg taattgctta 52860 tggagttgtc gtagttgctt ggagaaatgt cgtagttgga tggggagtag tcatagggaa 52920 gacgagcttc atccactaaa acaattggca ggtcagcaag tgcctgcccc gatgccatcg 52980 caagtacgag gcttagaacc accttcaaca gatcgcgcat agtcttcccc agctctctaa 53040 cgcttgagtt aagccgcgcc gcgaagcggc gtcggcttga acgaattgtt agacattatt 53100 tgccgactac cttggtgatc tcgcctttca cgtagtgaac aaattcttcc aactgatctg 53160 cgcgcgaggc caagcgatct tcttgtccaa gataagcctg cctagcttca agtatgacgg 53220 gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga 53280 ttttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat 53340 cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata 53400 gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc 53460 tatgttctct tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga 53520 agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg 53580 gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa 53640 tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg 53700 ttgtttcatc aagccttaca gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca 53760 ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc 53820 gctcgatgac gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc 53880 tcatgatgtt taactcctga attaagccgc gccgcgaagc ggtgtcggct tgaatgaatt 53940 gttaggcgtc atcctgtgct cccgagaacc agtaccagta catcgctgtt tcgttcgaga 54000 cttgaggtct agttttatac gtgaacaggt caatgccgcc gagagtaaag ccacattttg 54060 cgtacaaatt gcaggcaggt acattgttcg tttgtgtctc taatcgtatg ccaaggagct 54120 gtctgcttag tgcccacttt ttcgcaaatt cgatgagact gtgcgcgact cctttgcctc 54180 ggtgcgtgtg cgacacaaca atgtgttcga tagaggctag atcgttccat gttgagttga 54240 gttcaatctt cccgacaagc tcttggtcga tgaatgcgcc atagcaagca gagtcttcat 54300 cagagtcatc atccgagatg taatccttcc ggtaggggct cacacttctg gtagatagtt 54360 caaagccttg gtcggatagg tgcacatcga acacttcacg aacaatgaaa tggttctcag 54420 catccaatgt ttccgccacc tgctcaggga tcaccgaaat cttcatatga cgcctaacgc 54480 ctggcacagc ggatcgcaaa cctggcgcgg cttttggcac aaaaggcgtg acaggtttgc 54540 gaatccgttg ctgccacttg ttaacccttt tgccagattt ggtaactata atttatgtta 54600 gaggcgaagt cttgggtaaa aactggccta aaattgctgg ggatttcagg aaagtaaaca 54660 tcaccttccg gctcgatgtc tattgtagat atatgtagtg tatctacttg atcgggggat 54720 ctgctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 54780 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 54840 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt 54900 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg 54960 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc 55020 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 55080 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 55140 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 55200 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 55260 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 55320 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 55380 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 55440 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 55500 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 55560 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 55620 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 55680 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 55740 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 55800 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 55860 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 55920 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 55980

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   20161011_BB2533PCT_SeqLst.txt tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 56040 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 56100 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 56160 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 56220 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg gggggggggg 56280 gggggggact tccattgttc attccacgga caaaaacaga gaaaggaaac gacagaggcc 56340 aaaaagcctc gctttcagca cctgtcgttt cctttctttt cagagggtat tttaaataaa 56400 aacattaagt tatgacgaag aagaacggaa acgccttaaa ccggaaaatt ttcataaata 56460 gcgaaaaccc gcgaggtcgc cgccccgtaa cct 56493 <210> 47 <211> 55518 <212> DNA <213> Artificial sequence <220>

   <223> Agrobacterium vector containing maize codon optimized Cas9 and maize MDH promoter <400> 47 gtcggatcac cggaaaggac ccgtaaagtg gcgtggaggc catcaaacca cgtcaaataa atctgcatca acttaacgta aaaacaactt ggggcaacct catgtccccc cccccccccc gtttggtatg gcttcattca gctccggttc catgttgtgc aaaaaagcgg ttagctcctt ggccgcagtg ttatcactca tggttatggc atccgtaaga tgcttttctg tgactggtga tatgcggcga ccgagttgct cttgcccggc cagaacttta aaagtgctca tcattggaaa cttaccgctg ttgagatcca gttcgatgta atcttttact ttcaccagcg tttctgggtg aaagggaata agggcgacac ggaaatgttg ttgaagcatt tatcagggtt attgtctcat aaataaacaa ataggggttc cgcgcacatt aaccattatt atcatgacat taacctataa tcaagaattg gtcgacgatc ttgctgcgtt cccggattga aggcgagatc cagcaactcg gcgtgatgac tggccaggac gtcggccgaa gcgtcggatt tgcgatcgag gatttttcgg gatcaagcca cagcagccca ctcgaccttc ttggaatgct gctccgtcgt caggctttcc tcgtacggaa tgccaagcac tcccgagggg gacgaacgga taaacctttt cacgcccttt ttctcttagg tttacccgcc aatatatcct ggaaacgaca atctgatcat gagcggagaa tgacgcggga caagccgttt tacgtttgga ctcagcccaa gctggtacga ttgtaatacg aaacgctctt caactggaag agcggttacc tcgctatcaa ctttgtatag aaaagttggg cccggaccgg gttaccgaat tcgagctcgg gccgaagctt tttggaaggc taaggagagg tgtcactgtc ctgtcgtgtt ggctgttgac gaagatgcac attagcggcc tgaagtagag aactccccag acccgtaccc atgaacatag cctcgggtac gaaaatcctc ccatacccat tccatacccg acccgattat tcaaaaatta caatgcttgg gactctaggt ttttttactt tacaggccca aagttggtcg gcagccacta atgtcgttgg attgctggat ggtggaataa gggtattttt ctccatggct aatcgggttt atacccgatg ggtaagggat ttattccaaa taccttaacc ctaatagagg aattccccac ctctagactg aaggcgtcca actcaaatca ccggccgcac agcacaggct gcacagcccg ccagccaggt ccggcgtccg ggtctgcgcc cgatggtccc acatccatcc agcgggccgc aggtgcagct gcccaaacac cagacacaga ataatgatta tcatctacat atcacaacgt 60 tcaattatga cgcaggtatc gtattaattg 120 cagacaatac aaatcagcga cactgaatac 180 ccctgcaggc atcgtggtgt cacgctcgtc 240 ccaacgatca aggcgagtta catgatcccc 300 cggtcctccg atcgttgtca gaagtaagtt 360 agcactgcat aattctctta ctgtcatgcc 420 gtactcaacc aagtcattct gagaatagtg 480 gtcaacacgg gataataccg cgccacatag 540 acgttcttcg gggcgaaaac tctcaaggat 600 acccactcgt gcacccaact gatcttcagc 660 agcaaaaaca ggaaggcaaa atgccgcaaa 720 aatactcata ctcttccttt ttcaatatta 780 gagcggatac atatttgaat gtatttagaa 840 tccccgaaaa gtgccacctg acgtctaaga 900 aaataggcgt atcacgaggc cctttcgtct 960 cggatatttt cgtggagttc ccgccacaga 1020 cgccagatca tcctgtgacg gaactttggc 1080 agagcgacaa gcagatcacg cttttcgaca 1140 cgctgcgcta cgtccgcgac cgcgttgagg 1200 tagccgaccc agacgagcca agggatcttt 1260 gacgtttggg tggttgaaca gaagtcatta 1320 aaccctgtgg ttggcatgca catacaaatg 1380 taaatatccg ttattctaat aaacgctctt 1440 gtcaaacact gatagtttaa actgaaggcg 1500 ttaagggagt cacgttatga cccccgccga 1560 actgacagaa ccgcaacgtt gaaggagcca 1620 actcactata gggcgaattg agcgctgttt 1680 agaggccaga atggccatct cggaccgata 1740 ccgaattcga gctcggtacg gccagaatgg 1800 taccctggga tccgatatcg atgggccctg 1860 aagccggcga gaaggagggg gcgttttacg 1920 acgaatcatt tcttccgcgc gtgggaagaa 1980 atgtcaatgg ggaattcccc agcggggatt 2040 accggccccc atccccgaac ccgaacccga 2100 tcccgaccgg gtactaaata cccatgggta 2160 atgggctttt tatttgttaa ccggcggacg 2220 tgttgaccgg ctggcggctg ggctttttcc 2280 ggccacacgt cacaggcagc ccacaagtaa 2340 aaatcctaga tgctagattg ttctggttcc 2400 gggtttagcc ctcccaaacc cgaacccgcc 2460 tctataccca tggggatttg ttttaaccca 2520 gggtaatcgg gtttcggggc ccattgacat 2580 ttaaaaagtg ttgacgcacg cgctgatgcg 2640 tttaatcagc gatggagccc cggccgtcag 2700 ctgcggcgtc actgctgtcg ccaccgtctc 2760 gcgtggtaca aaaggctctt cctcgccgtc 2820 ctccaccacc ccgcttcgat cttctgttgc 2880

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   20161011_BB2533PCT_SeqLst.txt agctgaaatc tgtcagattc tgcagttcat tcctcatggc accgaagaag aagcgcaagg 2940 tgatggacaa gaagtacagc atcggcctcg acatcggcac caactcggtg ggctgggccg 3000 tcatcacgga cgaatataag gtcccgtcga agaagttcaa ggtcctcggc aatacagacc 3060 gccacagcat caagaaaaac ttgatcggcg ccctcctgtt cgatagcggc gagaccgcgg 3120 aggcgaccag gctcaagagg accgccagga gacggtacac taggcgcaag aacaggatct 3180 gctacctgca ggagatcttc agcaacgaga tggcgaaggt ggacgactcc ttcttccacc 3240 gcctggagga atcattcctg gtggaggagg acaagaagca tgagcggcac ccaatcttcg 3300 gcaacatcgt cgacgaggta agtttctgct tctacctttg atatatatat aataattatc 3360 attaattagt agtaatataa tatttcaaat atttttttca aaataaaaga atgtagtata 3420 tagcaattgc ttttctgtag tttataagtg tgtatatttt aatttataac ttttctaata 3480 tatgaccaaa acatggtgat gtgcaggtgg cctaccacga gaagtacccg acaatctacc 3540 acctccggaa gaaactggtg gacagcacag acaaggcgga cctccggctc atctaccttg 3600 ccctcgcgca tatgatcaag ttccgcggcc acttcctcat cgagggcgac ctgaacccgg 3660 acaactccga cgtggacaag ctgttcatcc agctcgtgca gacgtacaat caactgttcg 3720 aggagaaccc cataaacgct agcggcgtgg acgccaaggc catcctctcg gccaggctct 3780 cgaaatcaag aaggctggag aaccttatcg cgcagttgcc aggcgaaaag aagaacggcc 3840 tcttcggcaa ccttattgcg ctcagcctcg gcctgacgcc gaacttcaaa tcaaacttcg 3900 acctcgcgga ggacgccaag ctccagctct caaaggacac ctacgacgac gacctcgaca 3960 acctcctggc ccagatagga gaccagtacg cggacctctt cctcgccgcc aagaacctct 4020 ccgacgctat cctgctcagc gacatccttc gggtcaacac cgaaattacc aaggcaccgc 4080 tgtccgccag catgattaaa cgctacgacg agcaccatca ggacctcacg ctgctcaagg 4140 cactcgtccg ccagcagctc cccgagaagt acaaggagat cttcttcgac caatcaaaaa 4200 acggctacgc gggatatatc gacggcggtg ccagccagga agagttctac aagttcatca 4260 aaccaatcct ggagaagatg gacggcaccg aggagttgct ggtcaagctc aacagggagg 4320 acctcctcag gaagcagagg accttcgaca acggctccat cccgcatcag atccacctgg 4380 gcgaactgca tgccatcctg cggcgccagg aggacttcta cccgttcctg aaggataacc 4440 gggagaagat cgagaagatc ttgacgttcc gcatcccata ctacgtgggc ccgctggctc 4500 gcggcaactc ccggttcgcc tggatgaccc ggaagtcgga ggagaccatc acaccctgga 4560 actttgagga ggtggtcgat aagggcgcta gcgctcagag cttcatcgag cgcatgacca 4620 acttcgataa aaacctgccc aatgaaaaag tcctccccaa gcactcgctg ctctacgagt 4680 acttcaccgt gtacaacgag ctcaccaagg tcaaatacgt caccgagggc atgcggaagc 4740 cggcgttcct gagcggcgag cagaagaagg cgatagtgga cctcctcttc aagaccaaca 4800 ggaaggtgac cgtgaagcaa ttaaaagagg actacttcaa gaaaatagag tgcttcgact 4860 ccgtggagat ctcgggcgtg gaggatcggt tcaacgcctc actcggcacg tatcacgacc 4920 tcctcaagat cattaaagac aaggacttcc tcgacaacga ggagaacgag gacatcctcg 4980 aggacatcgt cctcaccctg accctgttcg aggaccgcga aatgatcgag gagaggctga 5040 agacctacgc gcacctgttc gacgacaagg tcatgaaaca gctcaagagg cgccgctaca 5100 ctggttgggg aaggctgtcc cgcaagctca ttaatggcat cagggacaag cagagcggca 5160 agaccatcct ggacttcctc aagtccgacg ggttcgccaa ccgcaacttc atgcagctca 5220 ttcacgacga ctcgctcacg ttcaaggaag acatccagaa ggcacaggtg agcgggcagg 5280 gtgactccct ccacgaacac atcgccaacc tggccggctc gccggccatt aaaaagggca 5340 tcctgcagac ggtcaaggtc gtcgacgagc tcgtgaaggt gatgggccgg cacaagcccg 5400 aaaatatcgt catagagatg gccagggaga accagaccac ccaaaaaggg cagaagaact 5460 cgcgcgagcg gatgaaacgg atcgaggagg gcattaaaga gctcgggtcc cagatcctga 5520 aggagcaccc cgtggaaaat acccagctcc agaatgaaaa gctctacctc tactacctgc 5580 agaacggccg cgacatgtac gtggaccagg agctggacat taatcggcta tcggactacg 5640 acgtcgacca catcgtgccg cagtcgttcc tcaaggacga tagcatcgac aacaaggtgc 5700 tcacccggtc ggataaaaat cggggcaaga gcgacaacgt gcccagcgag gaggtcgtga 5760 agaagatgaa aaactactgg cgccagctcc tcaacgcgaa actgatcacc cagcgcaagt 5820 tcgacaacct gacgaaggcg gaacgcggtg gcttgagcga actcgataag gcgggcttca 5880 taaaaaggca gctggtcgag acgcgccaga tcacgaagca tgtcgcccag atcctggaca 5940 gccgcatgaa tactaagtac gatgaaaacg acaagctgat ccgggaggtg aaggtgatca 6000 cgctgaagtc caagctcgtg tcggacttcc gcaaggactt ccagttctac aaggtccgcg 6060 agatcaacaa ctaccaccac gcccacgacg cctacctgaa tgcggtggtc gggaccgccc 6120 tgatcaagaa gtacccgaag ctggagtcgg agttcgtgta cggcgactac aaggtctacg 6180 acgtgcgcaa aatgatcgcc aagtccgagc aggagatcgg caaggccacg gcaaaatact 6240 tcttctactc gaacatcatg aacttcttca agaccgagat caccctcgcg aacggcgaga 6300 tccgcaagcg cccgctcatc gaaaccaacg gcgagacggg cgagatcgtc tgggataagg 6360 gccgggattt cgcgacggtc cgcaaggtgc tctccatgcc gcaagtcaat atcgtgaaaa 6420 agacggaggt ccagacgggc gggttcagca aggagtccat cctcccgaag cgcaactccg 6480 acaagctcat cgcgaggaag aaggattggg acccgaaaaa atatggcggc ttcgacagcc 6540 cgaccgtcgc atacagcgtc ctcgtcgtgg cgaaggtgga gaagggcaag tcaaagaagc 6600 tcaagtccgt gaaggagctg ctcgggatca cgattatgga gcggtcctcc ttcgagaaga 6660 acccgatcga cttcctagag gccaagggat ataaggaggt caagaaggac ctgattatta 6720 aactgccgaa gtactcgctc ttcgagctgg aaaacggccg caagaggatg ctcgcctccg 6780 caggcgagtt gcagaagggc aacgagctcg ccctcccgag caaatacgtc aatttcctgt 6840 acctcgctag ccactatgaa aagctcaagg gcagcccgga ggacaacgag cagaagcagc 6900 tcttcgtgga gcagcacaag cattacctgg acgagatcat cgagcagatc agcgagttct 6960

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   20161011_BB2533PCT_SeqLst.txt cgaagcgggt gatcctcgcc gacgcgaacc tggacaaggt gctgtcggca tataacaagc 7020 accgcgacaa accaatacgc gagcaggccg aaaatatcat ccacctcttc accctcacca 7080 acctcggcgc tccggcagcc ttcaagtact tcgacaccac gattgaccgg aagcggtaca 7140 cgagcacgaa ggaggtgctc gatgcgacgc tgatccacca gagcatcaca gggctctatg 7200 aaacacgcat cgacctgagc cagctgggcg gagacaagag accacgggac cgccacgatg 7260 gcgagctggg aggccgcaag cgggcaaggt aggtaccgtt aacctagact tgtccatctt 7320 ctggattggc caacttaatt aatgtatgaa ataaaaggat gcacacatag tgacatgcta 7380 atcactataa tgtgggcatc aaagttgtgt gttatgtgta attactagtt atctgaataa 7440 aagagaaaga gatcatccat atttcttatc ctaaatgaat gtcacgtgtc tttataattc 7500 tttgatgaac cagatgcatt tcattaacca aatccatata catataaata ttaatcatat 7560 ataattaata tcaattgggt tagcaaaaca aatctagtct aggtgtgttt tgcgaatgcg 7620 gccgccaccg cggtggagct cgaattcgag ctcggtaccc tgggatccag cttcgcttag 7680 tttttagttt ttggcagaaa aaatgatcaa tgtttcacaa accaaatatt tttataactt 7740 ttgatgaaag aagatcacca cggtcatatc taggggtggt aacaaattgc gatctaaatg 7800 tttcttcata aaaaataagg cttcttaata aattttagtt caaaataaat acgaataaag 7860 tctgattcta atctgattcg atccttaaat tttataatgc aaaatttaga gctcattacc 7920 acctctagtc atatgtctag tctgaggtat atccaaaaag ccctttctct aaattccaca 7980 cccaactcag atgtttgcaa ataaatactc cgactccaaa atgtaggtga agtgcaactt 8040 tctccatttt atatcaacat ttgttatttt ttgtttaaca tttcacactc aaaactaatt 8100 aataaaatac gtggttgttg aacgtgcgca catgtctccc ttacattatg tttttttatt 8160 tatgtattat tgttgttttc ctccgaacaa cttgtcaaca tatcatcatt ggtctttaat 8220 atttatgaat atggaagcct agttatttac acttggctac acactagttg tagttttgcc 8280 acttgtctaa catgcaactc tagtagtttt gccacttgcc tggcatgcaa ctctagtatt 8340 gacacttgta tagcatataa tgccaatacg acacctgcct tacatgaaac attatttttg 8400 acacttgtat accatgcaac attaccattg acatttgtcc atacacatta tatcaaatat 8460 attgagcgca tgtcacaaac tcgatacaaa gctggatgac cctccctcac cacatctata 8520 aaaacccgag cgctactgta aatcactcac aacacaacac atatctttta gtaacctttc 8580 aataggcgtc ccccaagaac tagtaaccat ggccctgtcc aacaagttca tcggcgacga 8640 catgaagatg acctaccaca tggacggctg cgtgaacggc cactacttca ccgtgaaggg 8700 cgagggcagc ggcaagccct acgagggcac ccagacctcc accttcaagg tgaccatggc 8760 caacggcggc cccctggcct tctccttcga catcctgtcc accgtgttca tgtacggcaa 8820 ccgctgcttc accgcctacc ccaccagcat gcccgactac ttcaagcagg ccttccccga 8880 cggcatgtcc tacgagagaa ccttcaccta cgaggacggc ggcgtggcca ccgccagctg 8940 ggagatcagc ctgaagggca actgcttcga gcacaagtcc accttccacg gcgtgaactt 9000 ccccgccgac ggccccgtga tggccaagaa gaccaccggc tgggacccct ccttcgagaa 9060 gatgaccgtg tgcgacggca tcttgaaggg cgacgtgacc gccttcctga tgctgcaggg 9120 cggcggcaac tacagatgcc agttccacac ctcctacaag accaagaagc ccgtgaccat 9180 gccccccaac cacgtggtgg agcaccgcat cgccagaacc gacctggaca agggcggcaa 9240 cagcgtgcag ctgaccgagc acgccgtggc ccacatcacc tccgtggtgc ccttctgaag 9300 cggcccatgg atattcgaac gcgtaggtac cacatggtta acctagactt gtccatcttc 9360 tggattggcc aacttaatta atgtatgaaa taaaaggatg cacacatagt gacatgctaa 9420 tcactataat gtgggcatca aagttgtgtg ttatgtgtaa ttactagtta tctgaataaa 9480 agagaaagag atcatccata tttcttatcc taaatgaatg tcacgtgtct ttataattct 9540 ttgatgaacc agatgcattt cattaaccaa atccatatac atataaatat taatcatata 9600 taattaatat caattgggtt agcaaaacaa atctagtcta ggtgtgtttt gcgaattccc 9660 atggacctcg agggggggcc cgggcaccca gctttcttgt acaaagtggc cgttaacgga 9720 tcggccagaa tggcccggac cgggttaccg aattcgagct cggtaccctg ggatcggccg 9780 ctctagaact agtggatccc ccgggctgca ggaattccca tggagtcaaa gattcaaata 9840 gaggacctaa cagaactcgc cgtaaagact ggcgaacagt tcatacagag tctcttacga 9900 ctcaatgaca agaagaaaat cttcgtcaac atggtggagc acgacacgct tgtctactcc 9960 aaaaatatca aagatacagt ctcagaagac caaagggcaa ttgagacttt tcaacaaagg 10020 gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag 10080 atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc 10140 gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc 10200 gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc 10260 actgacgtaa gggatgacgc acaatcccac taagcttcgg ccggggccca tcgatctggc 10320 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 10380 acgttgtaaa acgacggcca gtgccaagct cagatcagct tggggctggt atcgataaat 10440 gtttccacat agattttgca tatcataatg atgtttgtcg ttccgtatct atgtttcata 10500 caaaattttt acgcatatcg caacacatgg gcacatacct agtgactgta taactctgca 10560 tgtatgagtg tatgactata tgatgtagta actaataaga agggtagaca tttgagtgat 10620 tcttttattc ctggacttgt aagacttgac atttctgcct tgagtgcgat acatcatatg 10680 gacaggggtt atgcatacac tgcttgtttg ttgtttatgt tctaagagca tctccaacaa 10740 cgtgacatat gaaaatgccc tacaatttaa aaatggttat attttataaa atttagggca 10800 taaataaaac atcccgctcc aacattaaag ccttaaatct attataggga agcccactat 10860 gatatagtat atttgaggca ctttagaggg tgccctataa ttttttgacc atttttttat 10920 gaaatgagac actattggag tatttttttt ccgtagagca ccatatttca atttgagaca 10980 ccaatttaag gcattgttgg agatgttcta aatgttggtt tattttgtct gtatcgttgt 11040

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   20161011_BB2533PCT_SeqLst.txt ggttttgata gtggtgcctt tgcaatgtac atcttacatt gacaataata ataggtaaaa 11100 ctctacaaat tttttatcta atggactctt gtatgaaaca ttgtacttgc acacatctga 11160 tgtaaacact gcatactttt aacagtgaca agattctgtt tcattttagg gctagtttgg 11220 gaaccaaatt ttattagggt ttttattttc taagaaaaag taatttattt taccttgaga 11280 aaatataaat tacttgagaa aatagagttc caaactagct cttatctttg tcgaatcctc 11340 ctctattcaa atgtgacatt tctggcacgt gacaactggt gatgttgtag actgtgttaa 11400 gtaatacgtg tcattattac taaatgccat tttagtaaat gttgagtatg tactctacta 11460 cagtaagtat tattggtgta tttacactag acagttggcg gcctggcggg taaagttatc 11520 ctgtagaaag ttgggccagg ccaaaaccaa ccgccaaagg aaaggccttc cggcccgccc 11580 acctttgcgc gccgaaggtc agttccttca gtctcctccc gcttcagact ctgaccacgt 11640 cgacaatccg ggccgaaaca catctgcacc gtccacttgc gacagattga acacaccact 11700 tctatccacg tcagcgatcc gtggcactag cccttccacc aatcagccca agttgcccct 11760 ttcctttaaa ttcgccgcac ccattgctct tctcacggcc atagaaatcg accgagcgaa 11820 tccctcgcat cgcattcgca gcctttgctg catcacacca ccgcgaaacc ccagcagccg 11880 catctgcagg tcgactctag aggatccatg gcctcctccg aggacgtcat caaggagttc 11940 atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc acgagttcga gatcgagggc 12000 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc 12060 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg ctccaaggtg 12120 tacgtgaagc accccgccga catccccgac tacaagaagc tgtccttccc cgagggcttc 12180 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgacagtgac ccaggactcc 12240 tccctgcagg acggctcctt catctacaag gtgaagttca tcggcgtgaa cttcccctcc 12300 gacggccccg taatgcagaa gaagactatg ggctgggagg cctccaccga gcgcctgtac 12360 ccccgcgacg gcgtgctgaa gggcgagatc cacaaggccc tgaagctgaa ggacggcggc 12420 cacgctagcc catccaccca ctcactcact catatctgtg ctgtacgtac gagaatttct 12480 cgaccaaccg tcgtgagacc tgcccaccgg agatcggacg caagagggtt taggcaagaa 12540 tgtcgtgcga cagggtgagc gctgactagt atacgtgaga gaccttgaga tatacctcac 12600 acgtacgcgt actttacatg acgtaggaca ttacgactca aacagattca cgtcagattt 12660 cggagtttct cacgcgtgag agccttggag ggcggtatgt atgtcatact atatgttggg 12720 atggagggag tgagtgagtg atatgtggct agcaagggcg gccccctgcc cttcgcctgg 12780 gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac 12840 atccccgact acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac 12900 ttcgaggacg gcggcgtggt gacagtgacc caggactcct ccctgcagga cggctccttc 12960 atctacaagg tgaagttcat cggcgtgaac ttcccctccg acggccccgt aatgcagaag 13020 aagactatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag 13080 ggcgagatcc acaaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag 13140 tccatctaca tggccaagaa gcccgtgcag ctgcccggct actactacgt ggactccaag 13200 ctggacatca cctcccacaa cgaggactac accatcgtgg agcagtacga gcgcgccgag 13260 ggccgccacc acctgttcct gtagtcagga tctgagtcga aacctagact tgtccatctt 13320 ctggattggc caacttaatt aatgtatgaa ataaaaggat gcacacatag tgacatgcta 13380 atcactataa tgtgggcatc aaagttgtgt gttatgtgta attactagtt atctgaataa 13440 aagagaaaga gatcatccat atttcttatc ctaaatgaat gtcacgtgtc tttataattc 13500 tttgatgaac cagatgcatt tcattaacca aatccatata catataaata ttaatcatat 13560 ataattaata tcaattgggt tagcaaaaca aatctagtct aggtgtgttt tgcgaatgcg 13620 gccgccaccg cggtggagct cgaattccgg tccgggtcac ccggtccggg cctagaaggc 13680 cagcttgcgg ccgccccggg caactttatt atacaaagtt gatagatatc ggtccgagcg 13740 gcctagaagg cctttggtca cctttgtcca ccaagatgga actgcggccg ctcattaatt 13800 aagtcaggcg cgcctctagt tgaagacacg ttcatgtctt catcgtaaga agacactcag 13860 tagtcttcgg ccagaatggc ctaactcaag gccatcgtgg cctcttgctc ttcaggatga 13920 agagctatgt ttaaacgtgc aagcgctact agacaattca gtacattaaa aacgtccgca 13980 atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag 14040 ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc 14100 catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact ttgctcatgt 14160 taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac ggatgatctc 14220 gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg atcaaatatc 14280 atctccctcg cagagatccg aattatcagc cttcttattc atttctcgct taaccgtgac 14340 aggctgtcga tcttgagaac tatgccgaca taataggaaa tcgctggata aagccgctga 14400 ggaagctgag tggcgctatt tctttagaag tgaacgttga cgatcgtcga ccgtaccccg 14460 atgaattaat tcggacgtac gttctgaaca cagctggata cttacttggg cgattgtcat 14520 acatgacatc aacaatgtac ccgtttgtgt aaccgtctct tggaggttcg tatgacacta 14580 gtggttcccc tcagcttgcg actagatgtt gaggcctaac attttattag agagcaggct 14640 agttgcttag atacatgatc ttcaggccgt tatctgtcag ggcaagcgaa aattggccat 14700 ttatgacgac caatgccccg cagaagctcc catctttgcc gccatagacg ccgcgccccc 14760 cttttggggt gtagaacatc cttttgccag atgtggaaaa gaagttcgtt gtcccattgt 14820 tggcaatgac gtagtagccg gcgaaagtgc gagacccatt tgcgctatat ataagcctac 14880 gatttccgtt gcgactattg tcgtaattgg atgaactatt atcgtagttg ctctcagagt 14940 tgtcgtaatt tgatggacta ttgtcgtaat tgcttatgga gttgtcgtag ttgcttggag 15000 aaatgtcgta gttggatggg gagtagtcat agggaagacg agcttcatcc actaaaacaa 15060 ttggcaggtc agcaagtgcc tgccccgatg ccatcgcaag tacgaggctt agaaccacct 15120

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   20161011_BB2533PCT_SeqLst.txt tcaacagatc gcgcatagtc ttccccagct ctctaacgct tgagttaagc cgcgccgcga 15180 agcggcgtcg gcttgaacga attgttagac attatttgcc gactaccttg gtgatctcgc 15240 ctttcacgta gtgaacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt 15300 gtccaagata agcctgccta gcttcaagta tgacgggctg atactgggcc ggcaggcgct 15360 ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc 15420 aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg ggcggcgagt 15480 tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa 15540 agagttcctc cgccgctgga cctaccaagg caacgctatg ttctcttgct tttgtcagca 15600 agatagccag atcaatgtcg atcgtggctg gctcgaagat acctgcaaga atgtcattgc 15660 gctgccattc tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt 15720 cgtgcacaac aatggtgact tctacagcgc ggagaatctc gctctctcca ggggaagccg 15780 aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacagtca 15840 ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact gcggagccgt 15900 acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg 15960 atagttgagt cgatacttcg gcgatcaccg cttccctcat gatgtttaac tcctgaatta 16020 agccgcgccg cgaagcggtg tcggcttgaa tgaattgtta ggcgtcatcc tgtgctcccg 16080 agaaccagta ccagtacatc gctgtttcgt tcgagacttg aggtctagtt ttatacgtga 16140 acaggtcaat gccgccgaga gtaaagccac attttgcgta caaattgcag gcaggtacat 16200 tgttcgtttg tgtctctaat cgtatgccaa ggagctgtct gcttagtgcc cactttttcg 16260 caaattcgat gagactgtgc gcgactcctt tgcctcggtg cgtgtgcgac acaacaatgt 16320 gttcgataga ggctagatcg ttccatgttg agttgagttc aatcttcccg acaagctctt 16380 ggtcgatgaa tgcgccatag caagcagagt cttcatcaga gtcatcatcc gagatgtaat 16440 ccttccggta ggggctcaca cttctggtag atagttcaaa gccttggtcg gataggtgca 16500 catcgaacac ttcacgaaca atgaaatggt tctcagcatc caatgtttcc gccacctgct 16560 cagggatcac cgaaatcttc atatgacgcc taacgcctgg cacagcggat cgcaaacctg 16620 gcgcggcttt tggcacaaaa ggcgtgacag gtttgcgaat ccgttgctgc cacttgttaa 16680 cccttttgcc agatttggta actataattt atgttagagg cgaagtcttg ggtaaaaact 16740 ggcctaaaat tgctggggat ttcaggaaag taaacatcac cttccggctc gatgtctatt 16800 gtagatatat gtagtgtatc tacttgatcg ggggatctgc tgcctcgcgc gtttcggtga 16860 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 16920 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 16980 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca 17040 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 17100 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 17160 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 17220 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 17280 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 17340 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 17400 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 17460 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 17520 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 17580 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 17640 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 17700 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 17760 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 17820 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 17880 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 17940 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 18000 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 18060 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 18120 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 18180 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 18240 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 18300 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 18360 cgcaacgttg ttgccattgc tgcagggggg gggggggggg gggacttcca ttgttcattc 18420 cacggacaaa aacagagaaa ggaaacgaca gaggccaaaa agcctcgctt tcagcacctg 18480 tcgtttcctt tcttttcaga gggtatttta aataaaaaca ttaagttatg acgaagaaga 18540 acggaaacgc cttaaaccgg aaaattttca taaatagcga aaacccgcga ggtcgccgcc 18600 ccgtaacctg tcggatcacc ggaaaggacc cgtaaagtga taatgattat catctacata 18660 tcacaacgtg cgtggaggcc atcaaaccac gtcaaataat caattatgac gcaggtatcg 18720 tattaattga tctgcatcaa cttaacgtaa aaacaacttc agacaataca aatcagcgac 18780 actgaatacg gggcaacctc atgtcccccc cccccccccc cctgcaggca tcgtggtgtc 18840 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 18900 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 18960 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 19020 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 19080 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc 19140 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 19200

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   20161011_BB2533PCT_SeqLst.txt ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 19260 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 19320 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 19380 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 19440 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 19500 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 19560 ctttcgtctt caagaattcg gagcttttgc cattctcacc ggattcagtc gtcactcatg 19620 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg 19680 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg 19740 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg 19800 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta 19860 attggttgta acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat 19920 aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg 19980 ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat 20040 caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc 20100 agcaacacct tcttcacgag gcagacctca gcgccagaag gccgccagag aggccgagcg 20160 cggccgtgag gcttggacgc tagggcaggg catgaaaaag cccgtagcgg gctgctacgg 20220 gcgtctgacg cggtggaaag ggggagggga tgttgtctac atggctctgc tgtagtgagt 20280 gggttgcgct ccggcagcgg tcctgatcaa tcgtcaccct ttctcggtcc ttcaacgttc 20340 ctgacaacga gcctcctttt cgccaatcca tcgacaatca ccgcgagtcc ctgctcgaac 20400 gctgcgtccg gaccggcttc gtcgaaggcg tctatcgcgg cccgcaacag cggcgagagc 20460 ggagcctgtt caacggtgcc gccgcgctcg ccggcatcgc tgtcgccggc ctgctcctca 20520 agcacggccc caacagtgaa gtagctgatt gtcatcagcg cattgacggc gtccccggcc 20580 gaaaaacccg cctcgcagag gaagcgaagc tgcgcgtcgg ccgtttccat ctgcggtgcg 20640 cccggtcgcg tgccggcatg gatgcgcgcg ccatcgcggt aggcgagcag cgcctgcctg 20700 aagctgcggg cattcccgat cagaaatgag cgccagtcgt cgtcggctct cggcaccgaa 20760 tgcgtatgat tctccgccag catggcttcg gccagtgcgt cgagcagcgc ccgcttgttc 20820 ctgaagtgcc agtaaagcgc cggctgctga acccccaacc gttccgccag tttgcgtgtc 20880 gtcagaccgt ctacgccgac ctcgttcaac aggtccaggg cggcacggat cactgtattc 20940 ggctgcaact ttgtcatgct tgacacttta tcactgataa acataatatg tccaccaact 21000 tatcagtgat aaagaatccg cgcgttcaat cggaccagcg gaggctggtc cggaggccag 21060 acgtgaaacc caacataccc ctgatcgtaa ttctgagcac tgtcgcgctc gacgctgtcg 21120 gcatcggcct gattatgccg gtgctgccgg gcctcctgcg cgatctggtt cactcgaacg 21180 acgtcaccgc ccactatggc attctgctgg cgctgtatgc gttggtgcaa tttgcctgcg 21240 cacctgtgct gggcgcgctg tcggatcgtt tcgggcggcg gccaatcttg ctcgtctcgc 21300 tggccggcgc cactgtcgac tacgccatca tggcgacagc gcctttcctt tgggttctct 21360 atatcgggcg gatcgtggcc ggcatcaccg gggcgactgg ggcggtagcc ggcgcttata 21420 ttgccgatat cactgatggc gatgagcgcg cgcggcactt cggcttcatg agcgcctgtt 21480 tcgggttcgg gatggtcgcg ggacctgtgc tcggtgggct gatgggcggt ttctcccccc 21540 acgctccgtt cttcgccgcg gcagccttga acggcctcaa tttcctgacg ggctgtttcc 21600 ttttgccgga gtcgcacaaa ggcgaacgcc ggccgttacg ccgggaggct ctcaacccgc 21660 tcgcttcgtt ccggtgggcc cggggcatga ccgtcgtcgc cgccctgatg gcggtcttct 21720 tcatcatgca acttgtcgga caggtgccgg ccgcgctttg ggtcattttc ggcgaggatc 21780 gctttcactg ggacgcgacc acgatcggca tttcgcttgc cgcatttggc attctgcatt 21840 cactcgccca ggcaatgatc accggccctg tagccgcccg gctcggcgaa aggcgggcac 21900 tcatgctcgg aatgattgcc gacggcacag gctacatcct gcttgccttc gcgacacggg 21960 gatggatggc gttcccgatc atggtcctgc ttgcttcggg tggcatcgga atgccggcgc 22020 tgcaagcaat gttgtccagg caggtggatg aggaacgtca ggggcagctg caaggctcac 22080 tggcggcgct caccagcctg acctcgatcg tcggacccct cctcttcacg gcgatctatg 22140 cggcttctat aacaacgtgg aacgggtggg catggattgc aggcgctgcc ctctacttgc 22200 tctgcctgcc ggcgctgcgt cgcgggcttt ggagcggcgc agggcaacga gccgatcgct 22260 gatcgtggaa acgataggcc tatgccatgc gggtcaaggc gacttccggc aagctatacg 22320 cgccctagga gtgcggttgg aacgttggcc cagccagata ctcccgatca cgagcaggac 22380 gccgatgatt tgaagcgcac tcagcgtctg atccaagaac aaccatccta gcaacacggc 22440 ggtccccggg ctgagaaagc ccagtaagga aacaactgta ggttcgagtc gcgagatccc 22500 ccggaaccaa aggaagtagg ttaaacccgc tccgatcagg ccgagccacg ccaggccgag 22560 aacattggtt cctgtaggca tcgggattgg cggatcaaac actaaagcta ctggaacgag 22620 cagaagtcct ccggccgcca gttgccaggc ggtaaaggtg agcagaggca cgggaggttg 22680 ccacttgcgg gtcagcacgg ttccgaacgc catggaaacc gcccccgcca ggcccgctgc 22740 gacgccgaca ggatctagcg ctgcgtttgg tgtcaacacc aacagcgcca cgcccgcagt 22800 tccgcaaata gcccccagga ccgccatcaa tcgtatcggg ctacctagca gagcggcaga 22860 gatgaacacg accatcagcg gctgcacagc gcctaccgtc gccgcgaccc cgcccggcag 22920 gcggtagacc gaaataaaca acaagctcca gaatagcgaa atattaagtg cgccgaggat 22980 gaagatgcgc atccaccaga ttcccgttgg aatctgtcgg acgatcatca cgagcaataa 23040 acccgccggc aacgcccgca gcagcatacc ggcgacccct cggcctcgct gttcgggctc 23100 cacgaaaacg ccggacagat gcgccttgtg agcgtccttg gggccgtcct cctgtttgaa 23160 gaccgacagc ccaatgatct cgccgtcgat gtaggcgccg aatgccacgg catctcgcaa 23220 ccgttcagcg aacgcctcca tgggcttttt ctcctcgtgc tcgtaaacgg acccgaacat 23280

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   20161011_BB2533PCT_SeqLst.txt ctctggagct ttcttcaggg ccgacaatcg gatctcgcgg aaatcctgca cgtcggccgc 23340 tccaagccgt cgaatctgag ccttaatcac aattgtcaat tttaatcctc tgtttatcgg 23400 cagttcgtag agcgcgccgt gcgtcccgag cgatactgag cgaagcaagt gcgtcgagca 23460 gtgcccgctt gttcctgaaa tgccagtaaa gcgctggctg ctgaaccccc agccggaact 23520 gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca ggcgtgttcc 23580 accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc 23640 acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag cgggtacggc 23700 tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac 23760 ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg 23820 atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc 23880 agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt 23940 aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga ccagcccagg 24000 tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg 24060 tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc gacgccggtg 24120 taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc 24180 gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct 24240 cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc 24300 tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc tgacctgttt tgccaggtcc 24360 tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac 24420 ttcgccaaac ctgccgcctc ctgttcgaga cgacgcgaac gctccacggc ggccgatggc 24480 gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc 24540 tggaccatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt 24600 gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc 24660 ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgac tcacgccggg 24720 gcaatgtgcc cttattcctg atttgacccg cctggtgcct tggtgtccag ataatccacc 24780 ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt ccttctcgta cttggtattc 24840 cgaatcttgc cctgcacgaa taccagcgac cccttgccca aatacttgcc gtgggcctcg 24900 gcctgagagc caaaacactt gatgcggaag aagtcggtgc gctcctgctt gtcgccggca 24960 tcgttgcgcc actcttcatt aaccgctata tcgaaaattg cttgcggctt gttagaattg 25020 ccatgacgta cctcggtgtc acgggtaaga ttaccgataa actggaactg attatggctc 25080 atatcgaaag tctccttgag aaaggagact ctagtttagc taaacattgg ttccgctgtc 25140 aagaacttta gcggctaaaa ttttgcgggc cgcgaccaaa ggtgcgaggg gcggcttccg 25200 ctgtgtacaa ccagatattt ttcaccaaca tccttcgtct gctcgatgag cggggcatga 25260 cgaaacatga gctgtcggag agggcagggg tttcaatttc gtttttatca gacttaacca 25320 acggtaaggc caacccctcg ttgaaggtga tggaggccat tgccgacgcc ctggaaactc 25380 ccctacctct tctcctggag tccaccgacc ttgaccgcga ggcactcgcg gagattgcgg 25440 gtcatccttt caagagcagc gtgccgcccg gatacgaacg catcagtgtg gttttgccgt 25500 cacataaggc gtttatcgta aagaaatggg gcgacgacac ccgaaaaaag ctgcgtggaa 25560 ggctctgacg ccaagggtta gggcttgcac ttccttcttt agccgctaaa acggcccctt 25620 ctctgcgggc cgtcggctcg cgcatcatat cgacatcctc aacggaagcc gtgccgcgaa 25680 tggcatcggg cgggtgcgct ttgacagttg ttttctatca gaacccctac gtcgtgcggt 25740 tcgattagct gtttgtcttg caggctaaac actttcggta tatcgtttgc ctgtgcgata 25800 atgttgctaa tgatttgttg cgtaggggtt actgaaaagt gagcgggaaa gaagagtttc 25860 agaccatcaa ggagcgggcc aagcgcaagc tggaacgcga catgggtgcg gacctgttgg 25920 ccgcgctcaa cgacccgaaa accgttgaag tcatgctcaa cgcggacggc aaggtgtggc 25980 acgaacgcct tggcgagccg atgcggtaca tctgcgacat gcggcccagc cagtcgcagg 26040 cgattataga aacggtggcc ggattccacg gcaaagaggt cacgcggcat tcgcccatcc 26100 tggaaggcga gttccccttg gatggcagcc gctttgccgg ccaattgccg ccggtcgtgg 26160 ccgcgccaac ctttgcgatc cgcaagcgcg cggtcgccat cttcacgctg gaacagtacg 26220 tcgaggcggg catcatgacc cgcgagcaat acgaggtcat taaaagcgcc gtcgcggcgc 26280 atcgaaacat cctcgtcatt ggcggtactg gctcgggcaa gaccacgctc gtcaacgcga 26340 tcatcaatga aatggtcgcc ttcaacccgt ctgagcgcgt cgtcatcatc gaggacaccg 26400 gcgaaatcca gtgcgccgca gagaacgccg tccaatacca caccagcatc gacgtctcga 26460 tgacgctgct gctcaagaca acgctgcgta tgcgccccga ccgcatcctg gtcggtgagg 26520 tacgtggccc cgaagccctt gatctgttga tggcctggaa caccgggcat gaaggaggtg 26580 ccgccaccct gcacgcaaac aaccccaaag cgggcctgag ccggctcgcc atgcttatca 26640 gcatgcaccc ggattcaccg aaacccattg agccgctgat tggcgaggcg gttcatgtgg 26700 tcgtccatat cgccaggacc cctagcggcc gtcgagtgca agaaattctc gaagttcttg 26760 gttacgagaa cggccagtac atcaccaaaa ccctgtaagg agtatttcca atgacaacgg 26820 ctgttccgtt ccgtctgacc atgaatcgcg gcattttgtt ctaccttgcc gtgttcttcg 26880 ttctcgctct cgcgttatcc gcgcatccgg cgatggcctc ggaaggcacc ggcggcagct 26940 tgccatatga gagctggctg acgaacctgc gcaactccgt aaccggcccg gtggccttcg 27000 cgctgtccat catcggcatc gtcgtcgccg gcggcgtgct gatcttcggc ggcgaactca 27060 acgccttctt ccgaaccctg atcttcctgg ttctggtgat ggcgctgctg gtcggcgcgc 27120 agaacgtgat gagcaccttc ttcggtcgtg gtgccgaaat cgcggccctc ggcaacgggg 27180 cgctgcacca ggtgcaagtc gcggcggcgg atgccgtgcg tgcggtagcg gctggacggc 27240 tcgcctaatc atggctctgc gcacgatccc catccgtcgc gcaggcaacc gagaaaacct 27300 gttcatgggt ggtgatcgtg aactggtgat gttctcgggc ctgatggcgt ttgcgctgat 27360

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   20161011_BB2533PCT_SeqLst.txt tttcagcgcc caagagctgc gggccaccgt ggtcggtctg atcctgtggt tcggggcgct 27420 ctatgcgttc cgaatcatgg cgaaggccga tccgaagatg cggttcgtgt acctgcgtca 27480 ccgccggtac aagccgtatt acccggcccg ctcgaccccg ttccgcgaga acaccaatag 27540 ccaagggaag caataccgat gatccaagca attgcgattg caatcgcggg cctcggcgcg 27600 cttctgttgt tcatcctctt tgcccgcatc cgcgcggtcg atgccgaact gaaactgaaa 27660 aagcatcgtt ccaaggacgc cggcctggcc gatctgctca actacgccgc tgtcgtcgat 27720 gacggcgtaa tcgtgggcaa gaacggcagc tttatggctg cctggctgta caagggcgat 27780 gacaacgcaa gcagcaccga ccagcagcgc gaagtagtgt ccgcccgcat caaccaggcc 27840 ctcgcgggcc tgggaagtgg gtggatgatc catgtggacg ccgtgcggcg tcctgctccg 27900 aactacgcgg agcggggcct gtcggcgttc cctgaccgtc tgacggcagc gattgaagaa 27960 gagcgctcgg tcttgccttg ctcgtcggtg atgtacttca ccagctccgc gaagtcgctc 28020 ttcttgatgg agcgcatggg gacgtgcttg gcaatcacgc gcaccccccg gccgttttag 28080 cggctaaaaa agtcatggct ctgccctcgg gcggaccacg cccatcatga ccttgccaag 28140 ctcgtcctgc ttctcttcga tcttcgccag cagggcgagg atcgtggcat caccgaaccg 28200 cgccgtgcgc gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc ggcccaggtc 28260 gccattgatg cgggccagct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta 28320 gccctggccg acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc 28380 ctcaatcgct cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct 28440 ggttggcttg gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg 28500 ccagcctcgc agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa 28560 gaaggaacac ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg 28620 gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa 28680 ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa 28740 gcgctgcttc cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa 28800 attactgaac tgaggggaca ggcgagagac gatgccaaag agctacaccg acgagctggc 28860 cgagtgggtt gaatcccgcg cggccaagaa gcgccggcgt gatgaggctg cggttgcgtt 28920 cctggcggtg agggcggatg tcgaggcggc gttagcgtcc ggctatgcgc tcgtcaccat 28980 ttgggagcac atgcgggaaa cggggaaggt caagttctcc tacgagacgt tccgctcgca 29040 cgccaggcgg cacatcaagg ccaagcccgc cgatgtgccc gcaccgcagg ccaaggctgc 29100 ggaacccgcg ccggcaccca agacgccgga gccacggcgg ccgaagcagg ggggcaaggc 29160 tgaaaagccg gcccccgctg cggccccgac cggcttcacc ttcaacccaa caccggacaa 29220 aaaggatcta ctgtaatggc gaaaattcac atggttttgc agggcaaggg cggggtcggc 29280 aagtcggcca tcgccgcgat cattgcgcag tacaagatgg acaaggggca gacacccttg 29340 tgcatcgaca ccgacccggt gaacgcgacg ttcgagggct acaaggccct gaacgtccgc 29400 cggctgaaca tcatggccgg cgacgaaatt aactcgcgca acttcgacac cctggtcgag 29460 ctgattgcgc cgaccaagga tgacgtggtg atcgacaacg gtgccagctc gttcgtgcct 29520 ctgtcgcatt acctcatcag caaccaggtg ccggctctgc tgcaagaaat ggggcatgag 29580 ctggtcatcc ataccgtcgt caccggcggc caggctctcc tggacacggt gagcggcttc 29640 gcccagctcg ccagccagtt cccggccgaa gcgcttttcg tggtctggct gaacccgtat 29700 tgggggccta tcgagcatga gggcaagagc tttgagcaga tgaaggcgta cacggccaac 29760 aaggcccgcg tgtcgtccat catccagatt ccggccctca aggaagaaac ctacggccgc 29820 gatttcagcg acatgctgca agagcggctg acgttcgacc aggcgctggc cgatgaatcg 29880 ctcacgatca tgacgcggca acgcctcaag atcgtgcggc gcggcctgtt tgaacagctc 29940 gacgcggcgg ccgtgctatg agcgaccaga ttgaagagct gatccgggag attgcggcca 30000 agcacggcat cgccgtcggc cgcgacgacc cggtgctgat cctgcatacc atcaacgccc 30060 ggctcatggc cgacagtgcg gccaagcaag aggaaatcct tgccgcgttc aaggaagagc 30120 tggaagggat cgcccatcgt tggggcgagg acgccaaggc caaagcggag cggatgctga 30180 acgcggccct ggcggccagc aaggacgcaa tggcgaaggt aatgaaggac agcgccgcgc 30240 aggcggccga agcgatccgc agggaaatcg acgacggcct tggccgccag ctcgcggcca 30300 aggtcgcgga cgcgcggcgc gtggcgatga tgaacatgat cgccggcggc atggtgttgt 30360 tcgcggccgc cctggtggtg tgggcctcgt tatgaatcgc agaggcgcag atgaaaaagc 30420 ccggcgttgc cgggctttgt ttttgcgtta gctgggcttg tttgacaggc ccaagctctg 30480 actgcgcccg cgctcgcgct cctgggcctg tttcttctcc tgctcctgct tgcgcatcag 30540 ggcctggtgc cgtcgggctg cttcacgcat cgaatcccag tcgccggcca gctcgggatg 30600 ctccgcgcgc atcttgcgcg tcgccagttc ctcgatcttg ggcgcgtgaa tgcccatgcc 30660 ttccttgatt tcgcgcacca tgtccagccg cgtgtgcagg gtctgcaagc gggcttgctg 30720 ttgggcctgc tgctgctgcc aggcggcctt tgtacgcggc agggacagca agccgggggc 30780 attggactgt agctgctgca aacgcgcctg ctgacggtct acgagctgtt ctaggcggtc 30840 ctcgatgcgc tccacctggt catgctttgc ctgcacgtag agcgcaaggg tctgctggta 30900 ggtctgctcg atgggcgcgg attctaagag ggcctgctgt tccgtctcgg cctcctgggc 30960 cgcctgtagc aaatcctcgc cgctgttgcc gctggactgc tttactgccg gggactgctg 31020 ttgccctgct cgcgccgtcg tcgcagttcg gcttgccccc actcgattga ctgcttcatt 31080 tcgagccgca gcgatgcgat ctcggattgc gtcaacggac ggggcagcgc ggaggtgtcc 31140 ggcttctcct tgggtgagtc ggtcgatgcc atagccaaag gtttccttcc aaaatgcgtc 31200 cattgctgga ccgtgtttct cattgatgcc cgcaagcatc ttcggcttga ccgccaggtc 31260 aagcgcgcct tcatgggcgg tcatgacgga cgccgccatg accttgccgc cgttgttctc 31320 gatgtagccg cgtaatgagg caatggtgcc gcccatcgtc agcgtgtcat cgacaacgat 31380 gtacttctgg ccggggatca cctccccctc gaaagtcggg ttgaacgcca ggcgatgatc 31440

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   20161011_BB2533PCT_SeqLst.txt tgaaccggct ccggttcggg cgaccttctc ccgctgcaca atgtccgttt cgacctcaag 31500 gccaaggcgg tcggccagaa cgaccgccat catggccgga atcttgttgt tccccgccgc 31560 ctcgacggcg aggactggaa cgatgcgggg cttgtcgtcg ccgatcagcg tcttgagctg 31620 ggcaacagtg tcgtccgaaa tcaggcgctc gaccaaatta agcgccgctt ccgcgtcgcc 31680 ctgcttcgca gcctggtatt caggctcgtt ggtcaaagaa ccaaggtcgc cgttgcgaac 31740 caccttcggg aagtctcccc acggtgcgcg ctcggctctg ctgtagctgc tcaagacgcc 31800 tcccttttta gccgctaaaa ctctaacgag tgcgcccgcg actcaacttg acgctttcgg 31860 cacttacctg tgccttgcca cttgcgtcat aggtgatgct tttcgcactc ccgatttcag 31920 gtactttatc gaaatctgac cgggcgtgca ttacaaagtt cttccccacc tgttggtaaa 31980 tgctgccgct atctgcgtgg acgatgctgc cgtcgtggcg ctgcgactta tcggcctttt 32040 gggccatata gatgttgtaa atgccaggtt tcagggcccc ggctttatct accttctggt 32100 tcgtccatgc gccttggttc tcggtctgga caattctttg cccattcatg accaggaggc 32160 ggtgtttcat tgggtgactc ctgacggttg cctctggtgt taaacgtgtc ctggtcgctt 32220 gccggctaaa aaaaagccga cctcggcagt tcgaggccgg ctttccctag agccgggcgc 32280 gtcaaggttg ttccatctat tttagtgaac tgcgttcgat ttatcagtta ctttcctccc 32340 gctttgtgtt tcctcccact cgtttccgcg tctagccgac ccctcaacat agcggcctct 32400 tcttgggctg cctttgcctc ttgccgcgct tcgtcacgct cggcttgcac cgtcgtaaag 32460 cgctcggcct gcctggccgc ctcttgcgcc gccaacttcc tttgctcctg gtgggcctcg 32520 gcgtcggcct gcgccttcgc tttcaccgct gccaactccg tgcgcaaact ctccgcttcg 32580 cgcctggtgg cgtcgcgctc gccgcgaagc gcctgcattt cctggttggc cgcgtccagg 32640 gtcttgcggc tctcttcttt gaatgcgcgg gcgtcctggt gagcgtagtc cagctcggcg 32700 cgcagctcct gcgctcgacg ctccacctcg tcggcccgct gcgtcgccag cgcggcccgc 32760 tgctcggctc ctgccagggc ggtgcgtgct tcggccaggg cttgccgctg gcgtgcggcc 32820 agctcggccg cctcggcggc ctgctgctct agcaatgtaa cgcgcgcctg ggcttcttcc 32880 agctcgcggg cctgcgcctc gaaggcgtcg gccagctccc cgcgcacggc ttccaactcg 32940 ttgcgctcac gatcccagcc ggcttgcgct gcctgcaacg attcattggc aagggcctgg 33000 gcggcttgcc agagggcggc cacggcctgg ttgccggcct gctgcaccgc gtccggcacc 33060 tggactgcca gcggggcggc ctgcgccgtg cgctggcgtc gccattcgcg catgccggcg 33120 ctggcgtcgt tcatgttgac gcgggcggcc ttacgcactg catccacggt cgggaagttc 33180 tcccggtcgc cttgctcgaa cagctcgtcc gcagccgcaa aaatgcggtc gcgcgtctct 33240 ttgttcagtt ccatgttggc tccggtaatt ggtaagaata ataatactct tacctacctt 33300 atcagcgcaa gagtttagct gaacagttct cgacttaacg gcaggttttt tagcggctga 33360 agggcaggca aaaaaagccc cgcacggtcg gcgggggcaa agggtcagcg ggaaggggat 33420 tagcgggcgt cgggcttctt catgcgtcgg ggccgcgctt cttgggatgg agcacgacga 33480 agcgcgcacg cgcatcgtcc tcggccctat cggcccgcgt cgcggtcagg aacttgtcgc 33540 gcgctaggtc ctccctggtg ggcaccaggg gcatgaactc ggcctgctcg atgtaggtcc 33600 actccatgac cgcatcgcag tcgaggccgc gttccttcac cgtctcttgc aggtcgcggt 33660 acgcccgctc gttgagcggc tggtaacggg ccaattggtc gtaaatggct gtcggccatg 33720 agcggccttt cctgttgagc cagcagccga cgacgaagcc ggcaatgcag gcccctggca 33780 caaccaggcc gacgccgggg gcaggggatg gcagcagctc gccaaccagg aaccccgccg 33840 cgatgatgcc gatgccggtc aaccagccct tgaaactatc cggccccgaa acacccctgc 33900 gcattgcctg gatgctgcgc cggatagctt gcaacatcag gagccgtttc ttttgttcgt 33960 cagtcatggt ccgccctcac cagttgttcg tatcggtgtc ggacgaactg aaatcgcaag 34020 agctgccggt atcggtccag ccgctgtccg tgtcgctgct gccgaagcac ggcgaggggt 34080 ccgcgaacgc cgcagacggc gtatccggcc gcagcgcatc gcccagcatg gccccggtca 34140 gcgagccgcc ggccaggtag cccagcatgg tgctgttggt cgccccggcc accagggccg 34200 acgtgacgaa atcgccgtca ttccctctgg attgttcgct gctcggcggg gcagtgcgcc 34260 gcgccggcgg cgtcgtggat ggctcgggtt ggctggcctg cgacggccgg cgaaaggtgc 34320 gcagcagctc gttatcgacc ggctgcggcg tcggggccgc cgccttgcgc tgcggtcggt 34380 gttccttctt cggctcgcgc agcttgaaca gcatgatcgc ggaaaccagc agcaacgccg 34440 cgcctacgcc tcccgcgatg tagaacagca tcggattcat tcttcggtcc tccttgtagc 34500 ggaaccgttg tctgtgcggc gcgggtggcc cgcgccgctg tctttgggga tcagccctcg 34560 atgagcgcga ccagtttcac gtcggcaagg ttcgcctcga actcctggcc gtcgtcctcg 34620 tacttcaacc aggcatagcc ttccgccggc ggccgacggt tgaggataag gcgggcaggg 34680 cgctcgtcgt gctcgacctg gacgatggcc tttttcagct tgtccgggtc cggctccttc 34740 gcgccctttt ccttggcgtc cttaccgtcc tggtcgccgt cctcgccgtc ctggccgtcg 34800 ccggcctccg cgtcacgctc ggcatcagtc tggccgttga aggcatcgac ggtgttggga 34860 tcgcggccct tctcgtccag gaactcgcgc agcagcttga ccgtgccgcg cgtgatttcc 34920 tgggtgtcgt cgtcaagcca cgcctcgact tcctccgggc gcttcttgaa ggccgtcacc 34980 agctcgttca ccacggtcac gtcgcgcacg cggccggtgt tgaacgcatc ggcgatcttc 35040 tccggcaggt ccagcagcgt gacgtgctgg gtgatgaacg ccggcgactt gccgatttcc 35100 ttggcgatat cgcctttctt cttgcccttc gccagctcgc ggccaatgaa gtcggcaatt 35160 tcgcgcgggg tcagctcgtt gcgttgcagg ttctcgataa cctggtcggc ttcgttgtag 35220 tcgttgtcga tgaacgccgg gatggacttc ttgccggccc acttcgagcc acggtagcgg 35280 cgggcgccgt gattgatgat atagcggccc ggctgctcct ggttctcgcg caccgaaatg 35340 ggtgacttca ccccgcgctc tttgatcgtg gcaccgattt ccgcgatgct ctccggggaa 35400 aagccggggt tgtcggccgt ccgcggctga tgcggatctt cgtcgatcag gtccaggtcc 35460 agctcgatag ggccggaacc gccctgagac gccgcaggag cgtccaggag gctcgacagg 35520

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   20161011_BB2533PCT_SeqLst.txt tcgccgatgc tatccaaccc caggccggac ggctgcgccg cgcctgcggc ttcctgagcg 35580 gccgcagcgg tgtttttctt ggtggtcttg gcttgagccg cagtcattgg gaaatctcca 35640 tcttcgtgaa cacgtaatca gccagggcgc gaacctcttt cgatgccttg cgcgcggccg 35700 ttttcttgat cttccagacc ggcacaccgg atgcgagggc atcggcgatg ctgctgcgca 35760 ggccaacggt ggccggaatc atcatcttgg ggtacgcggc cagcagctcg gcttggtggc 35820 gcgcgtggcg cggattccgc gcatcgacct tgctgggcac catgccaagg aattgcagct 35880 tggcgttctt ctggcgcacg ttcgcaatgg tcgtgaccat cttcttgatg ccctggatgc 35940 tgtacgcctc aagctcgatg ggggacagca catagtcggc cgcgaagagg gcggccgcca 36000 ggccgacgcc aagggtcggg gccgtgtcga tcaggcacac gtcgaagcct tggttcgcca 36060 gggccttgat gttcgccccg aacagctcgc gggcgtcgtc cagcgacagc cgttcggcgt 36120 tcgccagtac cgggttggac tcgatgaggg cgaggcgcgc ggcctggccg tcgccggctg 36180 cgggtgcggt ttcggtccag ccgccggcag ggacagcgcc gaacagcttg cttgcatgca 36240 ggccggtagc aaagtccttg agcgtgtagg acgcattgcc ctgggggtcc aggtcgatca 36300 cggcaacccg caagccgcgc tcgaaaaagt cgaaggcaag atgcacaagg gtcgaagtct 36360 tgccgacgcc gcctttctgg ttggccgtga ccaaagtttt catcgtttgg tttcctgttt 36420 tttcttggcg tccgcttccc acttccggac gatgtacgcc tgatgttccg gcagaaccgc 36480 cgttacccgc gcgtacccct cgggcaagtt cttgtcctcg aacgcggccc acacgcgatg 36540 caccgcttgc gacactgcgc ccctggtcag tcccagcgac gttgcgaacg tcgcctgtgg 36600 cttcccatcg actaagacgc cccgcgctat ctcgatggtc tgctgcccca cttccagccc 36660 ctggatcgcc tcctggaact ggctttcggt aagccgtttc ttcatggata acacccataa 36720 tttgctccgc gccttggttg aacatagcgg tgacagccgc cagcacatga gagaagttta 36780 gctaaacatt tctcgcacgt caacaccttt agccgctaaa actcgtcctt ggcgtaacaa 36840 aacaaaagcc cggaaaccgg gctttcgtct cttgccgctt atggctctgc acccggctcc 36900 atcaccaaca ggtcgcgcac gcgcttcact cggttgcgga tcgacactgc cagcccaaca 36960 aagccggttg ccgccgccgc caggatcgcg ccgatgatgc cggccacacc ggccatcgcc 37020 caccaggtcg ccgccttccg gttccattcc tgctggtact gcttcgcaat gctggacctc 37080 ggctcaccat aggctgaccg ctcgatggcg tatgccgctt ctccccttgg cgtaaaaccc 37140 agcgccgcag gcggcattgc catgctgccc gccgctttcc cgaccacgac gcgcgcacca 37200 ggcttgcggt ccagaccttc ggccacggcg agctgcgcaa ggacataatc agccgccgac 37260 ttggctccac gcgcctcgat cagctcttgc actcgcgcga aatccttggc ctccacggcc 37320 gccatgaatc gcgcacgcgg cgaaggctcc gcagggccgg cgtcgtgatc gccgccgaga 37380 atgcccttca ccaagttcga cgacacgaaa atcatgctga cggctatcac catcatgcag 37440 acggatcgca cgaacccgct gaattgaaca cgagcacggc acccgcgacc actatgccaa 37500 gaatgcccaa ggtaaaaatt gccggccccg ccatgaagtc cgtgaatgcc ccgacggccg 37560 aagtgaaggg caggccgcca cccaggccgc cgccctcact gcccggcacc tggtcgctga 37620 atgtcgatgc cagcacctgc ggcacgtcaa tgcttccggg cgtcgcgctc gggctgatcg 37680 cccatcccgt tactgccccg atcccggcaa tggcaaggac tgccagcgct gccatttttg 37740 gggtgaggcc gttcgcggcc gaggggcgca gcccctgggg ggatgggagg cccgcgttag 37800 cgggccggga gggttcgaga agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac 37860 agggcgcagc cctggttaaa aacaaggttt ataaatattg gtttaaaagc aggttaaaag 37920 acaggttagc ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga ttttctgcct 37980 gtggacagcc cctcaaatgt caataggtgc gcccctcatc tgtcagcact ctgcccctca 38040 agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc 38100 agggcactta tccccaggct tgtccacatc atctgtggga aactcgcgta aaatcaggcg 38160 ttttcgccga tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg agcctgcccc 38220 tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat 38280 ctgtcagtga gggccaagtt ttccgcgagg tatccacaac gccggcggcc gcggtgtctc 38340 gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc cgccagccca 38400 gcggcgaggg caaccagccc ggtgagcgtc ggaaaggcgc tggaagcccc gtagcgacgc 38460 ggagaggggc gagacaagcc aagggcgcag gctcgatgcg cagcacgaca tagccggttc 38520 tcgcaaggac gagaatttcc ctgcggtgcc cctcaagtgt caatgaaagt ttccaacgcg 38580 agccattcgc gagagccttg agtccacgct agatgagagc tttgttgtag gtggaccagt 38640 tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga agatgcgtga 38700 tctgatcctt caactcagca aaagttcgat ttattcaaca aagccacgtt gtgtctcaaa 38760 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg 38820 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct 38880 cgactctaga gctcgttcct cgaggcctcg aggcctcgag gaacggtacc tgcggggaag 38940 cttacaataa tgtgtgttgt taagtcttgt tgcctgtcat cgtctgactg actttcgtca 39000 taaatcccgg cctccgtaac ccagctttgg gcaagctcac ggatttgatc cggcggaacg 39060 ggaatatcga gatgccgggc tgaacgctgc agttccagct ttccctttcg ggacaggtac 39120 tccagctgat tgattatctg ctgaagggtc ttggttccac ctcctggcac aatgcgaatg 39180 attacttgag cgcgatcggg catccaattt tctcccgtca ggtgcgtggt caagtgctac 39240 aaggcacctt tcagtaacga gcgaccgtcg atccgtcgcc gggatacgga caaaatggag 39300 cgcagtagtc catcgagggc ggcgaaagcc tcgccaaaag caatacgttc atctcgcaca 39360 gcctccagat ccgatcgagg gtcttcggcg taggcagata gaagcatgga tacattgctt 39420 gagagtattc cgatggactg aagtatggct tccatctttt ctcgtgtgtc tgcatctatt 39480 tcgagaaagc ccccgatgcg gcgcaccgca acgcgaattg ccatactatc cgaaagtccc 39540 agcaggcgcg cttgatagga aaaggtttca tactcggccg atcgcagacg ggcactcacg 39600

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   20161011_BB2533PCT_SeqLst.txt accttgaacc cttcaacttt cagggatcga tgctggttga tggtagtctc actcgacgtg 39660 gctctggtgt gttttgacat agcttcctcc aaagaaagcg gaaggtctgg atactccagc 39720 acgaaatgtg cccgggtaga cggatggaag tctagccctg ctcaatatga aatcaacagt 39780 acatttacag tcaatactga atatacttgc tacatttgca attgtcttat aacgaatgtg 39840 aaataaaaat agtgtaacaa cgcttttact catcgataat cacaaaaaca tttatacgaa 39900 caaaaataca aatgcactcc ggtttcacag gataggcggg atcagaatat gcaacttttg 39960 acgttttgtt ctttcaaagg gggtgctggc aaaaccaccg cactcatggg cctttgcgct 40020 gctttggcaa atgacggtaa acgagtggcc ctctttgatg ccgacgaaaa ccggcctctg 40080 acgcgatgga gagaaaacgc cttacaaagc agtactggga tcctcgctgt gaagtctatt 40140 ccgccgacga aatgcccctt cttgaagcag cctatgaaaa tgccgagctc gaaggatttg 40200 attatgcgtt ggccgatacg cgtggcggct cgagcgagct caacaacaca atcatcgcta 40260 gctcaaacct gcttctgatc cccaccatgc taacgccgct cgacatcgat gaggcactat 40320 ctacctaccg ctacgtcatc gagctgctgt tgagtgaaaa tttggcaatt cctacagctg 40380 ttttgcgcca acgcgtcccg gtcggccgat tgacaacatc gcaacgcagg atgtcagaga 40440 cgctagagag ccttccagtt gtaccgtctc ccatgcatga aagagatgca tttgccgcga 40500 tgaaagaacg cggcatgttg catcttacat tactaaacac gggaactgat ccgacgatgc 40560 gcctcataga gaggaatctt cggattgcga tggaggaagt cgtggtcatt tcgaaactga 40620 tcagcaaaat cttggaggct tgaagatggc aattcgcaag cccgcattgt cggtcggcga 40680 agcacggcgg cttgctggtg ctcgacccga gatccaccat cccaacccga cacttgttcc 40740 ccagaagctg gacctccagc acttgcctga aaaagccgac gagaaagacc agcaacgtga 40800 gcctctcgtc gccgatcaca tttacagtcc cgatcgacaa cttaagctaa ctgtggatgc 40860 ccttagtcca cctccgtccc cgaaaaagct ccaggttttt ctttcagcgc gaccgcccgc 40920 gcctcaagtg tcgaaaacat atgacaacct cgttcggcaa tacagtccct cgaagtcgct 40980 acaaatgatt ttaaggcgcg cgttggacga tttcgaaagc atgctggcag atggatcatt 41040 tcgcgtggcc ccgaaaagtt atccgatccc ttcaactaca gaaaaatccg ttctcgttca 41100 gacctcacgc atgttcccgg ttgcgttgct cgaggtcgct cgaagtcatt ttgatccgtt 41160 ggggttggag accgctcgag ctttcggcca caagctggct accgccgcgc tcgcgtcatt 41220 ctttgctgga gagaagccat cgagcaattg gtgaagaggg acctatcgga acccctcacc 41280 aaatattgag tgtaggtttg aggccgctgg ccgcgtcctc agtcaccttt tgagccagat 41340 aattaagagc caaatgcaat tggctcaggc tgccatcgtc cccccgtgcg aaacctgcac 41400 gtccgcgtca aagaaataac cggcacctct tgctgttttt atcagttgag ggcttgacgg 41460 atccgcctca agtttgcggc gcagccgcaa aatgagaaca tctatactcc tgtcgtaaac 41520 ctcctcgtcg cgtactcgac tggcaatgag aagttgctcg cgcgatagaa cgtcgcgggg 41580 tttctctaaa aacgcgagga gaagattgaa ctcacctgcc gtaagtttca cctcaccgcc 41640 agcttcggac atcaagcgac gttgcctgag attaagtgtc cagtcagtaa aacaaaaaga 41700 ccgtcggtct ttggagcgga caacgttggg gcgcacgcgc aaggcaaccc gaatgcgtgc 41760 aagaaactct ctcgtactaa acggcttagc gataaaatca cttgctccta gctcgagtgc 41820 aacaacttta tccgtctcct caaggcggtc gccactgata attatgattg gaatatcaga 41880 ctttgccgcc agatttcgaa cgatctcaag cccatcttca cgacctaaat ttagatcaac 41940 aaccacgaca tcgaccgtcg cggaagagag tactctagtg aactgggtgc tgtcggctac 42000 cgcggtcact ttgaaggcgt ggatcgtaag gtattcgata ataagatgcc gcatagcgac 42060 atcgtcatcg ataagaagaa cgtgtttcaa cggctcacct ttcaatctaa aatctgaacc 42120 cttgttcaca gcgcttgaga aattttcacg tgaaggatgt acaatcatct ccagctaaat 42180 gggcagttcg tcagaattgc ggctgaccgc ggatgacgaa aatgcgaacc aagtatttca 42240 attttatgac aaaagttctc aatcgttgtt acaagtgaaa cgcttcgagg ttacagctac 42300 tattgattaa ggagatcgcc tatggtctcg ccccggcgtc gtgcgtccgc cgcgagccag 42360 atctcgccta cttcataaac gtcctcatag gcacggaatg gaatgatgac atcgatcgcc 42420 gtagagagca tgtcaatcag tgtgcgatct tccaagctag caccttgggc gctacttttg 42480 acaagggaaa acagtttctt gaatccttgg attggattcg cgccgtgtat tgttgaaatc 42540 gatcccggat gtcccgagac gacttcactc agataagccc atgctgcatc gtcgcgcatc 42600 tcgccaagca atatccggtc cggccgcata cgcagacttg cttggagcaa gtgctcggcg 42660 ctcacagcac ccagcccagc accgttcttg gagtagagta gtctaacatg attatcgtgt 42720 ggaatgacga gttcgagcgt atcttctatg gtgattagcc tttcctgggg ggggatggcg 42780 ctgatcaagg tcttgctcat tgttgtcttg ccgcttccgg tagggccaca tagcaacatc 42840 gtcagtcggc tgacgacgca tgcgtgcaga aacgcttcca aatccccgtt gtcaaaatgc 42900 tgaaggatag cttcatcatc ctgattttgg cgtttccttc gtgtctgcca ctggttccac 42960 ctcgaagcat cataacggga ggagacttct ttaagaccag aaacacgcga gcttggccgt 43020 cgaatggtca agctgacggt gcccgaggga acggtcggcg gcagacagat ttgtagtcgt 43080 tcaccaccag gaagttcagt ggcgcagagg gggttacgtg gtccgacatc ctgctttctc 43140 agcgcgcccg ctaaaatagc gatatcttca agatcatcat aagagacggg caaaggcatc 43200 ttggtaaaaa tgccggcttg gcgcacaaat gcctctccag gtcgattgat cgcaatttct 43260 tcagtcttcg ggtcatcgag ccattccaaa atcggcttca gaagaaagcg tagttgcgga 43320 tccacttcca tttacaatgt atcctatctc taagcggaaa tttgaattca ttaagagcgg 43380 cggttcctcc cccgcgtggc gccgccagtc aggcggagct ggtaaacacc aaagaaatcg 43440 aggtcccgtg ctacgaaaat ggaaacggtg tcaccctgat tcttcttcag ggttggcggt 43500 atgttgatgg ttgccttaag ggctgtctca gttgtctgct caccgttatt ttgaaagctg 43560 ttgaagctca tcccgccacc cgagctgccg gcgtaggtgc tagctgcctg gaaggcgcct 43620 tgaacaacac tcaagagcat agctccgcta aaacgctgcc agaagtggct gtcgaccgag 43680

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   20161011_BB2533PCT_SeqLst.txt cccggcaatc ctgagcgacc gagttcgtcc gcgcttggcg atgttaacga gatcatcgca 43740 tggtcaggtg tctcggcgcg atcccacaac acaaaaacgc gcccatctcc ctgttgcaag 43800 ccacgctgta tttcgccaac aacggtggtg ccacgatcaa gaagcacgat attgttcgtt 43860 gttccacgaa tatcctgagg caagacacac tttacatagc ctgccaaatt tgtgtcgatt 43920 gcggtttgca agatgcacgg aattattgtc ccttgcgtta ccataaaatc ggggtgcggc 43980 aagagcgtgg cgctgctggg ctgcagctcg gtgggtttca tacgtatcga caaatcgttc 44040 tcgccggaca cttcgccatt cggcaaggag ttgtcgtcac gcttgccttc ttgtcttcgg 44100 cccgtgtcgc cctgaatggc gcgtttgctg accccttgat cgccgctgct atatgcaaaa 44160 atcggtgttt cttccggccg tggctcatgc cgctccggtt cgcccctcgg cggtagagga 44220 gcagcaggct gaacagcctc ttgaaccgct ggaggatccg gcggcacctc aatcggagct 44280 ggatgaaatg gcttggtgtt tgttgcgatc aaagttgacg gcgatgcgtt ctcattcacc 44340 ttcttttggc gcccacctag ccaaatgagg cttaatgata acgcgagaac gacacctccg 44400 acgatcaatt tctgagaccc cgaaagacgc cggcgatgtt tgtcggagac cagggatcca 44460 gatgcatcaa cctcatgtgc cgcttgctga ctatcgttat tcatcccttc gcccccttca 44520 ggacgcgttt cacatcgggc ctcaccgtgc ccgtttgcgg cctttggcca acgggatcgt 44580 aagcggtgtt ccagatacat agtactgtgt ggccatccct cagacgccaa cctcgggaaa 44640 ccgaagaaat ctcgacatcg ctccctttaa ctgaatagtt ggcaacagct tccttgccat 44700 caggattgat ggtgtagatg gagggtatgc gtacattgcc cggaaagtgg aataccgtcg 44760 taaatccatt gtcgaagact tcgagtggca acagcgaacg atcgccttgg gcgacgtagt 44820 gccaattact gtccgccgca ccaagggctg tgacaggctg atccaataaa ttctcagctt 44880 tccgttgata ttgtgcttcc gcgtgtagtc tgtccacaac agccttctgt tgtgcctccc 44940 ttcgccgagc cgccgcatcg tcggcggggt aggcgaattg gacgctgtaa tagagatcgg 45000 gctgctcttt atcgaggtgg gacagagtct tggaacttat actgaaaaca taacggcgca 45060 tcccggagtc gcttgcggtt agcacgatta ctggctgagg cgtgaggacc tggcttgcct 45120 tgaaaaatag ataatttccc cgcggtaggg ctgctagatc tttgctattt gaaacggcaa 45180 ccgctgtcac cgtttcgttc gtggcgaatg ttacgaccaa agtagctcca accgccgtcg 45240 agaggcgcac cacttgatcg ggattgtaag ccaaataacg catgcgcgga tctagcttgc 45300 ccgccattgg agtgtcttca gcctccgcac cagtcgcagc ggcaaataaa catgctaaaa 45360 tgaaaagtgc ttttctgatc atggttcgct gtggcctacg tttgaaacgg tatcttccga 45420 tgtctgatag gaggtgacaa ccagacctgc cgggttggtt agtctcaatc tgccgggcaa 45480 gctggtcacc ttttcgtagc gaactgtcgc ggtccacgta ctcaccacag gcattttgcc 45540 gtcaacgacg agggtccttt tatagcgaat ttgctgcgtg cttggagtta catcatttga 45600 agcgatgtgc tcgacctcca ccctgccgcg tttgccaaga atgacttgag gcgaactggg 45660 attgggatag ttgaagaatt gctggtaatc ctggcgcact gttggggcac tgaagttcga 45720 taccaggtcg taggcgtact gagcggtgtc ggcatcataa ctctcgcgca ggcgaacgta 45780 ctcccacaat gaggcgttaa cgacggcctc ctcttgagtt gcaggcaatc gcgagacaga 45840 cacctcgctg tcaacggtgc cgtccggccg tatccataga tatacgggca caagcctgct 45900 caacggcacc attgtggcta tagcgaacgc ttgagcaaca tttcccaaaa tcgcgatagc 45960 tgcgacagct gcaatgagtt tggagagacg tcgcgccgat ttcgctcgcg cggtttgaaa 46020 ggcttctact tccttatagt gctcggcaag gctttcgcgc gccactagca tggcatattc 46080 aggccccgtc atagcgtcca cccgaattgc cgagctgaag atctgacgga gtaggctgcc 46140 atcgccccac attcagcggg aagatcgggc ctttgcagct cgctaatgtg tcgtttgtct 46200 ggcagccgct caaagcgaca actaggcaca gcaggcaata cttcatagaa ttctccattg 46260 aggcgaattt ttgcgcgacc tagcctcgct caacctgagc gaagcgacgg tacaagctgc 46320 tggcagattg ggttgcgccg ctccagtaac tgcctccaat gttgccggcg atcgccggca 46380 aagcgacaat gagcgcatcc cctgtcagaa aaaacatatc gagttcgtaa agaccaatga 46440 tcttggccgc ggtcgtaccg gcgaaggtga ttacaccaag cataagggtg agcgcagtcg 46500 cttcggttag gatgacgatc gttgccacga ggtttaagag gagaagcaag agaccgtagg 46560 tgataagttg cccgatccac ttagctgcga tgtcccgcgt gcgatcaaaa atatatccga 46620 cgaggatcag aggcccgatc gcgagaagca ctttcgtgag aattccaacg gcgtcgtaaa 46680 ctccgaaggc agaccagagc gtgccgtaaa ggacccactg tgccccttgg aaagcaagga 46740 tgtcctggtc gttcatcgga ccgatttcgg atgcgatttt ctgaaaaacg gcctgggtca 46800 cggcgaacat tgtatccaac tgtgccggaa cagtctgcag aggcaagccg gttacactaa 46860 actgctgaac aaagtttggg accgtctttt cgaagatgga aaccacatag tcttggtagt 46920 tagcctgccc aacaattaga gcaacaacga tggtgaccgt gatcacccga gtgataccgc 46980 tacgggtatc gacttcgccg cgtatgacta aaataccctg aacaataatc caaagagtga 47040 cacaggcgat caatggcgca ctcaccgcct cctggatagt ctcaagcatc gagtccaagc 47100 ctgtcgtgaa ggctacatcg aagatcgtat gaatggccgt aaacggcgcc ggaatcgtga 47160 aattcatcga ttggacctga acttgactgg tttgtcgcat aatgttggat aaaatgagct 47220 cgcattcggc gaggatgcgg gcggatgaac aaatcgccca gccttagggg agggcaccaa 47280 agatgacagc ggtcttttga tgctccttgc gttgagcggc cgcctcttcc gcctcgtgaa 47340 ggccggcctg cgcggtagtc atcgttaata ggcttgtcgc ctgtacattt tgaatcattg 47400 cgtcatggat ctgcttgaga agcaaaccat tggtcacggt tgcctgcatg atattgcgag 47460 atcgggaaag ctgagcagac gtatcagcat tcgccgtcaa gcgtttgtcc atcgtttcca 47520 gattgtcagc cgcaatgcca gcgctgtttg cggaaccggt gatctgcgat cgcaacaggt 47580 ccgcttcagc atcactaccc acgactgcac gatctgtatc gctggtgatc gcacgtgccg 47640 tggtcgacat tggcattcgc ggcgaaaaca tttcattgtc taggtccttc gtcgaaggat 47700 actgattttt ctggttgagc gaagtcagta gtccagtaac gccgtaggcc gacgtcaaca 47760

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   20161011_BB2533PCT_SeqLst.txt tcgtaaccat cgctatagtc tgagtgagat tctccgcagt cgcgagcgca gtcgcgagcg 47820 tctcagcctc cgttgccggg tcgctaacaa caaactgcgc ccgcgcgggc tgaatatata 47880 gaaagctgca ggtcaaaact gttgcaataa gttgcgtcgt cttcatcgtt tcctacctta 47940 tcaatcttct gcctcgtggt gacgggccat gaattcgctg agccagccag atgagttgcc 48000 ttcttgtgcc tcgcgtagtc gagttgcaaa gcgcaccgtg ttggcacgcc ccgaaagcac 48060 ggcgacatat tcacgcatat cccgcagatc aaattcgcag atgacgcttc cactttctcg 48120 tttaagaaga aacttacggc tgccgaccgt catgtcttca cggatcgcct gaaattcctt 48180 ttcggtacat ttcagtccat cgacataagc cgatcgatct gcggttggtg atggatagaa 48240 aatcttcgtc atacattgcg caaccaagct ggctcctagc ggcgattcca gaacatgctc 48300 tggttgctgc gttgccagta ttagcatccc gttgtttttt cgaacggtca ggaggaattt 48360 gtcgacgaca gtcgaaaatt tagggtttaa caaataggcg cgaaactcat cgcagctcat 48420 cacaaaacgg cggccgtcga tcatggctcc aatccgatgc aggagatatg ctgcagcggg 48480 agcgcatact tcctcgtatt cgagaagatg cgtcatgtcg aagccggtaa tcgacggatc 48540 taactttact tcgtcaactt cgccgtcaaa tgcccagcca agcgcatggc cccggcacca 48600 gcgttggagc cgcgctcctg cgccttcggc gggcccatgc aacaaaaatt cacgtaaccc 48660 cgcgattgaa cgcatttgtg gatcaaacga gagctgacga tggataccac ggaccagacg 48720 gcggttctct tccggagaaa tcccaccccg accatcactc tcgatgagag ccacgatcca 48780 ttcgcgcaga aaatcgtgtg aggctgctgt gttttctagg ccacgcaacg gcgccaaccc 48840 gctgggtgtg cctctgtgaa gtgccaaata tgttcctcct gtggcgcgaa ccagcaattc 48900 gccaccccgg tccttgtcaa agaacacgac cgtacctgca cggtcgacca tgctctgttc 48960 gagcatggct agaacaaaca tcatgagcgt cgtcttaccc ctcccgatag gcccgaatat 49020 tgccgtcatg ccaacatcgt gctcatgcgg gatatagtcg aaaggcgttc cgccattggt 49080 acgaaatcgg gcaatcgcgt tgccccagtg gcctgagctg gcgccctctg gaaagttttc 49140 gaaagagaca aaccctgcga aattgcgtga agtgattgcg ccagggcgtg tgcgccactt 49200 aaaattcccc ggcaattggg accaataggc cgcttccata ccaatacctt cttggacaac 49260 cacggcacct gcatccgcca ttcgtgtccg agcccgcgcg cccctgtccc caagactatt 49320 gagatcgtct gcatagacgc aaaggctcaa atgatgtgag cccataacga attcgttgct 49380 cgcaagtgcg tcctcagcct cggataattt gccgatttga gtcacggctt tatcgccgga 49440 actcagcatc tggctcgatt tgaggctaag tttcgcgtgc gcttgcgggc gagtcaggaa 49500 cgaaaaactc tgcgtgagaa caagtggaaa atcgagggat agcagcgcgt tgagcatgcc 49560 cggccgtgtt tttgcagggt attcgcgaaa cgaatagatg gatccaacgt aactgtcttt 49620 tggcgttctg atctcgagtc ctcgcttgcc gcaaatgact ctgtcggtat aaatcgaagc 49680 gccgagtgag ccgctgacga ccggaaccgg tgtgaaccga ccagtcatga tcaaccgtag 49740 cgcttcgcca atttcggtga agagcacacc ctgcttctcg cggatgccaa gacgatgcag 49800 gccatacgct ttaagagagc cagcgacaac atgccaaaga tcttccatgt tcctgatctg 49860 gcccgtgaga tcgttttccc tttttccgct tagcttggtg aacctcctct ttaccttccc 49920 taaagccgcc tgtgggtaga caatcaacgt aaggaagtgt tcattgcgga ggagttggcc 49980 ggagagcacg cgctgttcaa aagcttcgtt caggctagcg gcgaaaacac tacggaagtg 50040 tcgcggcgcc gatgatggca cgtcggcatg acgtacgagg tgagcatata ttgacacatg 50100 atcatcagcg atattgcgca acagcgtgtt gaacgcacga caacgcgcat tgcgcatttc 50160 agtttcctca agctcgaatg caacgccatc aattctcgca atggtcatga tcgatccgtc 50220 ttcaagaagg acgatatggt cgctgaggtg gccaatataa gggagataga tctcaccgga 50280 tctttcggtc gttccactcg cgccgagcat cacaccattc ctctccctcg tgggggaacc 50340 ctaattggat ttgggctaac agtagcgccc ccccaaactg cactatcaat gcttcttccc 50400 gcggtccgca aaaatagcag gacgacgctc gccgcattgt agtctcgctc cacgatgagc 50460 cgggctgcaa accataacgg cacgagaacg acttcgtaga gcgggttctg aacgataacg 50520 atgacaaagc cggcgaacat catgaataac cctgccaatg tcagtggcac cccaagaaac 50580 aatgcgggcc gtgtggctgc gaggtaaagg gtcgattctt ccaaacgatc agccatcaac 50640 taccgccagt gagcgtttgg ccgaggaagc tcgccccaaa catgataaca atgccgccga 50700 cgacgccggc aaccagccca agcgaagccc gcccgaacat ccaggagatc ccgatagcga 50760 caatgccgag aacagcgagt gactggccga acggaccaag gataaacgtg catatattgt 50820 taaccattgt ggcggggtca gtgccgccac ccgcagattg cgctgcggcg ggtccggatg 50880 aggaaatgct ccatgcaatt gcaccgcaca agcttggggc gcagctcgat atcacgcgca 50940 tcatcgcatt cgagagcgag aggcgattta gatgtaaacg gtatctctca aagcatcgca 51000 tcaatgcgca cctccttagt ataagtcgaa taagacttga ttgtcgtctg cggatttgcc 51060 gttgtcctgg tgtggcggtg gcggagcgat taaaccgcca gcgccatcct cctgcgagcg 51120 gcgctgatat gacccccaaa catcccacgt ctcttcggat tttagcgcct cgtgatcgtc 51180 ttttggaggc tcgattaacg cgggcaccag cgattgagca gctgtttcaa cttttcgcac 51240 gtagccgttt gcaaaaccgc cgatgaaatt accggtgttg taagcggaga tcgcccgacg 51300 aagcgcaaat tgcttctcgt caatcgtttc gccgcctgca taacgacttt tcagcatgtt 51360 tgcagcggca gataatgatg tgcacgcctg gagcgcaccg tcaggtgtca gaccgagcat 51420 agaaaaattt cgagagttta tttgcatgag gccaacatcc agcgaatgcc gtgcatcgag 51480 acggtgcctg acgacttggg ttgcttggct gtgatcttgc cagtgaagcg tttcgccggt 51540 cgtgttgtca tgaatcgcta aaggatcaaa gcgactctcc accttagcta tcgccgcaag 51600 cgtagatgtc gcaactgatg gggcacactt gcgagcaaca tggtcaaact cagcagatga 51660 gagtggcgtg gcaaggctcg acgaacagaa ggagaccatc aaggcaagag aaagcgaccc 51720 cgatctctta agcatacctt atctccttag ctcgcaacta acaccgcctc tcccgttgga 51780 agaagtgcgt tgttttatgt tgaagattat cgggagggtc ggttactcga aaattttcaa 51840

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   20161011_BB2533PCT_SeqLst.txt ttgcttcttt atgatttcaa ttgaagcgag aaacctcgcc cggcgtcttg gaacgcaaca 51900 tggaccgaga accgcgcatc catgactaag caaccggatc gacctattca ggccgcagtt 51960 ggtcaggtca ggctcagaac gaaaatgctc ggcgaggtta cgctgtctgt aaacccattc 52020 gatgaacggg aagcttcctt ccgattgctc ttggcaggaa tattggccca tgcctgcttg 52080 cgctttgcaa atgctcttat cgcgttggta tcatatgcct tgtccgccag cagaaacgca 52140 ctctaagcga ttatttgtaa aaatgtttcg gtcatgcggc ggtcatgggc ttgacccgct 52200 gtcagcgcaa gacggatcgg tcaaccgtcg gcatcgacaa cagcgtgaat cttggtggtc 52260 aaaccgccac gggaacgtcc catacagcca tcgtcttgat cccgctgttt cccgtcgccg 52320 catgttggtg gacgcggaca caggaactgt caatcatgac gacattctat cgaaagcctt 52380 ggaaatcaca ctcagaatat gatcccagac gtctgcctca cgccatcgta caaagcgatt 52440 gtagcaggtt gtacaggaac cgtatcgatc aggaacgtct gcccagggcg ggcccgtccg 52500 gaagcgccac aagatgacat tgatcacccg cgtcaacgcg cggcacgcga cgcggcttat 52560 ttgggaacaa aggactgaac aacagtccat tcgaaatcgg tgacatcaaa gcggggacgg 52620 gttatcagtg gcctccaagt caagcctcaa tgaatcaaaa tcagaccgat ttgcaaacct 52680 gatttatgag tgtgcggcct aaatgatgaa atcgtccttc tagatcgcct ccgtggtgta 52740 gcaacacctc gcagtatcgc cgtgctgacc ttggccaggg aattgactgg caagggtgct 52800 ttcacatgac cgctcttttg gccgcgatag atgatttcgt tgctgctttg ggcacgtaga 52860 aggagagaag tcatatcgga gaaattcctc ctggcgcgag agcctgctct atcgcgacgg 52920 catcccactg tcgggaacag accggatcat tcacgaggcg aaagtcgtca acacatgcgt 52980 tataggcatc ttcccttgaa ggatgatctt gttgctgcca atctggaggt gcggcagccg 53040 caggcagatg cgatctcagc gcaacttgcg gcaaaacatc tcactcacct gaaaaccact 53100 agcgagtctc gcgatcagac gaaggccttt tacttaacga cacaatatcc gatgtctgca 53160 tcacaggcgt cgctatccca gtcaatacta aagcggtgca ggaactaaag attactgatg 53220 acttaggcgt gccacgaggc ctgagacgac gcgcgtagac agttttttga aatcattatc 53280 aaagtgatgg cctccgctga agcctatcac ctctgcgccg gtctgtcgga gagatgggca 53340 agcattatta cggtcttcgc gcccgtacat gcattggacg attgcagggt caatggatct 53400 gagatcatcc agaggattgc cgcccttacc ttccgtttcg agttggagcc agcccctaaa 53460 tgagacgaca tagtcgactt gatgtgacaa tgccaagaga gagatttgct taacccgatt 53520 tttttgctca agcgtaagcc tattgaagct tgccggcatg acgtccgcgc cgaaagaata 53580 tcctacaagt aaaacattct gcacaccgaa atgcttggtg tagacatcga ttatgtgacc 53640 aagatcctta gcagtttcgc ttggggaccg ctccgaccag aaataccgaa gtgaactgac 53700 gccaatgaca ggaatccctt ccgtctgcag ataggtacca tcgatagatc tgctgcctcg 53760 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 53820 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 53880 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct 53940 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 54000 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga 54060 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 54120 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 54180 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 54240 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 54300 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 54360 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 54420 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 54480 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 54540 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 54600 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 54660 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 54720 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 54780 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 54840 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 54900 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 54960 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 55020 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 55080 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 55140 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 55200 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 55260 agttaatagt ttgcgcaacg ttgttgccat tgctgcaggg gggggggggg gggggttcca 55320 ttgttcattc cacggacaaa aacagagaaa ggaaacgaca gaggccaaaa agctcgcttt 55380 cagcacctgt cgtttccttt cttttcagag ggtattttaa ataaaaacat taagttatga 55440 cgaagaagaa cggaaacgcc ttaaaccgga aaattttcat aaatagcgaa aacccgcgag 55500 gtcgccgccc cgtaacct 55518 <210> 48 <211> 61 <212> DNA <213> artificial sequence

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   20161011_BB2533PCT_SeqLst.txt <220>

   <223> reference sequence <400> 48

   cacgtatata tacgcgtacg cgtacgtgtg c aggtatatat atcctccgcc ggggcacgta 60 61 <210> 49 <211> 62 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 49 cacgtatata tacgcgtacg cgtacgttgt gaggtatata tatcctccgc cggggcacgt 60 ac 62 <210> 50 <211> 60 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 50 cacgtatata tacgcgtacg cgtacggtga ggtatatata tcctccgccg gggcacgtac 60 <210> 51 <211> 60 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 51 cacgtatata tacgcgtacg cgtactgtga ggtatatata tcctccgccg gggcacgtac 60 <210> 52 <211> 59 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 52 cacgtatata tacgcgtacg cgtacgtgag gtatatatat cctccgccgg ggcacgtac 59 <210> 53 <211> 32 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 53 cacgtatata tatcctccgc cggggcacgt ac 32 <210> 54 <211> 19 <212> DNA <213> artificial sequence

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   20161011_BB2533PCT_SeqLst.txt <220>

   <223> sequence on Fig. 1 <400> 54 cacgtatata tacgcgtac 19 <210> 55 <211> 57 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 1 <400> 55

   cacgtatata tacgcgtacg cgtgtgaggt atatatatcc tccgccgggg cacgtac 57 <210> <211> <212> <213> 56 33 DNA artificial sequence <220> <223> sequence on Fig. 1 <400> 56 cacgtatata tacgcgtacg ccggggcacg tac 33 <210> 57 <211> 30 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 57 cacgtatata tcctccgccg gggcacgtac 30 <210> 58 <211> 59 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 1 <400> 58 cacgtatata tacgcgtacg cgtatgtgag gtatatatat cctccgccgg ggcacgtac 59 <210> 59 <211> 54 <212> DNA <213> artificial sequence <220> <223> sequence on Fig. 2 <400> 59 gcgctgctcg attccgtccc catggtcgcc atcacgggac aggtgccgcg acgc 54

   <210> 60 <211> 18 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 2

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   20161011_BB2533PCT_SeqLst.txt <400> 60

   Ala Leu Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Pro 1 5 10 15

   Arg Arg <210> 61 <211> 54 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 2 <400> 61 gcgttgctcg actccgtccc cattgtcgcc atcacgggac aggtgtcgcg acgc <210> 62 <211> 18 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 2 <400> 62

   Ala Leu Leu Asp Ser Val Pro Ile Val Ala Ile Thr Gly Gln Val Ser 1 5 10 15

   Arg Arg <210> 63 <211> 54 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 2 <400> 63 gcgttgctgg actccgtgcc gatggtcgcc atcacgggac aggtgtcccg acgc <210> 64 <211> 18 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 2 <400> 64

   Ala Leu Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Ser 1 5 10 15

   Arg Arg <210> 65 <211> 31 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 4 <400> 65 atggctcccc cggccacccc gctccggccg t

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   20161011_BB2533PCT_SeqLst.txt <210> 66 <211> 10 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 4 <400> 66

   Met Ala Pro Pro Ala Thr Pro Leu Arg Pro

   1 5 10 <210> 67 <211> 30 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 4 <400> 67 atggctcccc cggccacccc ctccggccgt 30 <210> 68 <211> 10 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 4 <400> 68

   Met Ala Pro Pro Ala Thr Pro Ser Gly Arg

   1 5 10 <210> 69 <211> 31 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 4 <220>

   <221> misc_feature <222> (21)..(21) <223> n is a, c, g, or t <400> 69 atggctcccc cggccacccc nctccggccg t 31 <210> 70 <211> 40 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 5 <400> 70 tcgactcgct caccatgtcc ggcccatgac caccgccgcc 40 <210> 71 <211> 41 <212> DNA <213> artificial sequence <220>

   Page 45

   20161011_BB2533PCT_SeqLst.txt <223> sequence on Fig. 5 <220>

   <221> misc_feature <222> (17)..(17) <223> n is a, c, g, or t <400> 71 tcgactcgct caccatngtc cggcccatga ctcccccggc c 41 <210> 72 <211> 24 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 6 <400> 72 attcccccgg ccaccccgtc ggcc 24 <210> 73 <211> 8 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 6 <400> 73

   Ile Pro Pro Ala Thr Pro Ser Ala

   1 5 <210> 74 <211> 23 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 6 <400> 74 attcccccgg ccacccgtcg gcc 23 <210> 75 <211> 7 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 6 <400> 75

   Ile Pro Pro Ala Thr Arg Arg

   1 5 <210> 76 <211> 24 <212> DNA <213> artificial sequence <220>

   <223> sequence on Fig. 6 <400> 76 attcccccgg ccacctcgtc ggcc 24 <210> 77

   Page 46

   20161011_BB2533PCT_SeqLst.txt <211> 8 <212> PRT <213> artificial sequence <220>

   <223> sequence on Fig. 6 <400> 77

   Ile Pro Pro Ala Thr Ser Ser Ala 1 5 <210> 78 <211> 441 <212> DNA <213> Artificial Sequence

   <220> <223> IN2 promoter <400> 78 atccctggcc accaaacatc cctaatcatc cccaaatttt ataggaacta ctaatttctc 60 taacttaaaa aaaatctaaa atagtatact ttagcagcct ctcaatctga tttgttcccc 120 aaatttgaat cctggcttcg ctctgtcacc tgttgtactc tacatggtgc gcagggggag 180 agcctaatct ttcacgactt tgtttgtaac tgttagccag accggcgtat ttgtcaatgt 240 ataaacacgt aataaaattt acgtaccata tagtaagact ttgtatataa gacgtcacct 300 cttacgtgca tggttatatg cgacatgtgc agtgacgtta tcagatatag ctcaccctat 360 atatatagct ctgtccggtg tcagtagcaa tcaccattca tcagcacccc ggcaggtcga 420 ccccgagctc cctgcacctg c 441 <210> 79 <211> 21 <212> DNA <213> Zea mays <400> 79 gctcccccgg ccaccccgct c 21 <210> 80 <211> 20 <212> DNA <213> Zea mays <400> 80 gctcccccgg ccaccccctc 20 <210> 81 <211> 20 <212> DNA <213> Zea mays <400> 81 cgccgagggc gactaccggc 20 <210> 82 <211> 23 <212> DNA <213> Zea mays <400> 82 cgccgagggc gactaccggc agg 23

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